Sarah A Scott

Oscar Cordero-Llana

Sarah L Maslen

Jane M Anderson

Julia Boyle

Ruma-Raha Chowhdury

Pam Tyers

Roger A Barker

Claire M Kelly

Anne E Rosser

Elaine Stephens

Siddharthan Chandran

Maeve A Caldwell
email: maeve.caldwell@bristol.ac.uk

Clusterin secreted by astrocytes enhances neuronal differentiation from human neural precursor cells Running Title : Clusterin enhances neuronal differentiation

Neuronal differentiation from expanded human ventral mesencephalic neural precursor cells (NPCs) is very limited. Astrocytes are known to secrete neurotrophic factors and so in order to enhance neuronal survival from NPCs, we tested the effect of regional astrocyte conditioned medium (ACM) from the rat cortex, hippocampus and midbrain on this process. Human NPC's were expanded in FGF-2 prior to differentiation for 1 or 4 weeks in ACM. Results show that ACM from the hippocampus and midbrain increase the number of neurons from expanded human NPCs, an effect that was not observed with cortical ACM. In addition, both hippocampal and midbrain ACM increased the number and length of phosphorylated neurofilaments (pNF). Maldi-TOF analysis used to determine differences in media revealed that although all three regional ACMs had cystatin C, α-2 macroglobulin, extracellular matrix glycoprotein and vimentin, only hippocampal and midbrain ACM also contained clusterin, which when immunodepleted from midbrain ACM eliminated the observed effects on neuronal differentiation. Furthermore, clusterin is a highly glycosylated protein that has no effect on cell proliferation but decreases apoptotic nuclei and causes a sustained increase in pERK, implicating its role in cell survival and differentiation. These findings further reveal differential effects of regional astrocytes on NPC behaviour and identify clusterin as an important mediator of NPC derived neuronal survival and differentiation.

Introduction

Astrocytes are essential regulators of neuronal function and produce diffusible and non-diffusible neuron supporting signals, including neurotrophic factors and membrane-bound molecules (for reviews see 1, 2). Interestingly, emerging evidence highlights that there are many regional differences in astrocytes in respect to structure and function and soluble factor production, for example astrocytes from the hippocampus and their conditioned media can support the survival of neurons from regions other than the hippocampus [START_REF] Yoshida | Neurotropic effect of conditioned media of astrocytes isolated from different brain regions on hippocampal and cortical neurons[END_REF][START_REF] Hewett | Determinants of regional and local diversity within the astroglial lineage of the normal central nervous system[END_REF]. In contrast, astrocytes from the mesencephalon release factors, which are more effective at promoting dopamine neuron survival than cortical or striatal astrocytes [START_REF] Takeshima | Astrocyte dependent and independent phases of the development and survival of rat embryonic day 14 mesencephalic, dopaminergic neurons in culture[END_REF]. These studies demonstrate that astrocytes from neurogenic regions [START_REF] Suhonen | Differentiation of adult hippocampus derived progenitors into olfactory neurons in vivo[END_REF] release more neurogenic factors than astrocytes from non-neurogenic regions.

It is well established that stem cells can be isolated from the developing and adult rodent CNS and expanded in vitro using EGF and FGF2 (for review see 7) More recently the same has been shown to be true for the developing human fetal CNS [START_REF] Carpenter | In vitro expansion of a multipotent population of human neural progenitor cells[END_REF].

These cells are capable of long-term expansion in vitro to achieve approximately 50 population doublings [START_REF] Svendsen | A new method for the rapid and long term growth of human neural precursor cells[END_REF]. However, as they progress in culture with time their differentiation pattern changes with the number of neurons declining and the number of astrocytes predominating [START_REF] Ostenfeld | Regional specification of rodent and human neurospheres[END_REF][START_REF] Jain | GABAergi immunoreactivity is predominant in neurons derived from expanded neural precursor cells in vitro[END_REF][START_REF] Zietlow | The survival of neural precursor cell grafts is influenced by in vivo expansion[END_REF].

Previous studies have shown that astrocyte condition media (ACM) increases the number of neurons from adult rodent neural stem cells [START_REF] Song | Gage FH Astroglia induce neurogenesis from adult neural stem cell[END_REF]. The aim of this present study was to further explore the neurogenic inducing properties of astrocytes by investigating if ACM from cortical, hippocampal or midbrain origins had any effect on or 1 month in control media or in regional ACM. Results show that hippocampal and midbrain ACM increase the number of TuJ1 + and SMI312 + neurons. Mass spectrometric analysis revealed that clusterin is secreted by astrocytes from the midbrain and hippocampus, and subsequent immunodepletion experiments showed that this secreted protein is likely to be responsible for the increase in neuronal differentiation from NPCs. In addition, clusterin is a highly glycosylated protein that decreases TUNEL positive nuclei and causes a sustained activation of ERK phosphorylation, indicating its role in cell survival and differentiation.

METHODS

Preparation of astrocyte-conditioned medium

Mixed glial cultures from P0 rat pups were prepared as previously described [START_REF] Mccarthy | deVellis J Preparation of separate astriglial and oligodendroglial cell cultures from rat cerebral tissue[END_REF][START_REF] Wilkins | Oligodendrocytes promote neuronal survival and axonal length by distinct intracellular mechanisms: a novel role for oligodendrocyte derived glial cell line derived neurotropic factor[END_REF]. Culture medium used was DMEM, 1% penicillin G/streptomycin/amphotericin and 10% Fetal Calf Serum (FCS). Medium was changed after 24h and twice weekly thereafter until the cells reached confluence (8-10 days). Microglia were removed by vigorous shaking of the cultures at 240 rpm (20 min), followed by removal of oligodendrocyte progenitor cells by shaking at 160 rpm (overnight). The remaining monolayer was treated with 20μM Ara-C for 72h, and washed with DMEM / 1% PSF / 1% N2. 24h later fresh DMEM / 1% PSF was added for a further 48h. Media was then filtered, aliquoted and stored at -20°C. To enable the ACM to be analysed on the mass spectrometer, DMEM was replaced with Phenol free DMEM and supplemented with glucose (3500mg/l) and L-glutamine (4mM).

Human neurosphere Cell Culture and Immunohistochemistry

Human ventral mesencephalon (VM) or cortex (8-10 weeks post-conception: n=6) was collected following routine terminations of pregnancies. The methods of collection conformed to arrangements recommended by the Polkinghorne Committee (1989) and the UK Department of Health guidelines (1995). Human neurospheres were expanded as previously described [START_REF] Svendsen | A new method for the rapid and long term growth of human neural precursor cells[END_REF]. To induce differentiation, neurospheres were plated on poly-D-lysine/laminin in DMEM, 1% N2, 1μg/ml Bovine serum albumin, 0.6μg/ml N-Acetyl-cysteine, 5nM forskolin. ACM from the cortex, hippocampus or midbrain made up 50% of the media. Half the media was replaced every 4d. 0.2 μM BrdU was added to the medium on plating day for 7d. After 7d or 28d the cells were fixed in PFA (4%). Primary and secondary antibodies are detailed in Suppl Table1. TUNEL cell death assays were carried out following manufacturer's guidelines (Roche).

Imaging and quantification of Cultured Cells.

Cultures were viewed under a Leitz DMRD fluorescent microscope attached to a Hamamatsu C4742-95 digital camera and visualised using Openlab 2.1 (Improvision, Coventry, UK). Images were taken from a minimum of 4 random fields per coverslip from 4 coverslips per culture (x20 magnification) from at least 4 independent cultures.

Results were analysed by One-way ANOVA followed by Newman-keuls post-hoc test.

MALDI Tandem Mass Spectrometric Analysis and protein identification

Proteins in 200 μl of cell media were precipitated with acetone, resolubilised in 0.3% sodium dodecyl sulphate (SDS), reduced and alkylated with S-methyl methanethiosulfonate and digested with trypsin (Promega) at 37 ºC overnight. The peptide mixture was zip-tipped (C18; Waters) and analysed by nanoLC-MALDI as previously described [START_REF] Stephens | The N-linked oligosaccharides of aminopeptidase N from Manduca sexta: site localization and identification of novel N-glycan structures[END_REF]. Mass spectrometric analysis was achieved using a MALDI-TOF/TOF instrument (4700 Proteomics Analyzer, Applied Biosystems).

Mass Spectrometric Data Analysis. The MS/MS spectra were searched against all species in the National Center for Biotechnology Information (NCBI) database using GPS software (Applied Biosystems) running Mascot search algorithm (Matrix Science, Boston, MA) for peptide and protein identification. A mass tolerance of 100ppm and 0.25Da was used for precursors and fragment ions, respectively. The data searches were performed with the following criteria: variable modifications with methionine oxidation, S-methyl methanethiosulfonate derivation (MMTS) of cysteine and N-terminal pyroglutamate. Two missed cleavages were allowed. At least 95% confidence interval threshold was used for peptide identification and manual validation was also performed for each MS/MS spectrum.

Immunodepletion of clusterin from midbrain astrocytes conditioned medium.

SDS-gel electrophoresis and Western blot analysis.

After electrophoretical separation of protein extracts in 10% SDS-Tris-Glycine gels, proteins were blotted onto 0.45 μm PVDF membranes. Membranes were blocked with 5% non-fat dry 8 milk and 0.05% Tween20 (Sigma-Aldrich, Munich, Germany) in TBS-buffer (BioRad, Munich, Germany). After incubation with primary antibody (anti-Rat Clusterin, 1: 100 in blocking buffer) (rabbit polyclonal Antibody BioVendor Laboratory Medicine, Inc) and HRPlabelled secondary antibody anti-rabbit IgG (1:3000; DAKO; in blocking buffer) protein bands were visualized with ECL plus chemiluminescence developing agent (Amersham ECL plus, GE Healthcare, UK).

Semi-automated Image Acquisition and Analysis

Results

ACM from the hippocampus and midbrain, but not cortex increase the number of neurons from human NPCs.

In order to determine if regional ACM had an effect on the number of NPC derived neurons, human VM was expanded for 4 weeks in FGF2 prior to plating in control, cortical, hippocampal or midbrain ACM. Results demonstrate that midbrain ACM increased the number of neurons after a week and month of differentiation (P<0.05; Interestingly cortical ACM had no effect on neurogenic potential. We next examined ACMs effects on morphology and specifically on neurofilament phosphorylation using SMI312. This antibody is specific for phospho-epitopes in neurofilaments and was used as a marker for axonal integrity. Results show that midbrain ACM significantly increased the number of SMI312+ neurons (P<0.001; Fig 1A ,1B) after a week (P<0.01) and a month (P<0.01). However, hippocampal ACM showed an increase after a month only (P<0.05; Fig 1A). In order to determine if regional ACM had an effect on neurite length, neurite lengths per SMI312+ neurons were measured. Both hippocampal and midbrain ACM significantly increased neurite length (Figure 1A).

Again cortical ACM showed no effect. Cultures were also stained for the astrocyte marker GFAP and none of the ACMs had an effect on astrocyte numbers (Figure 2) revealing a neuronal specific effect for the ACM.

Protein identification by mass spectroscopy

In order to determine the major protein components secreted by the regional astrocytes, nanoLC-MALDI-TOF/TOF-MS/MS analyses were carried out on peptides produced from tryptic digestion of proteins in the ACM. ACM was collected in a minimal phenol free media containing high glucose and glutamine that did not contain N2 and B27 cell media supplements, which are known to contain significant amounts of potentially confounding proteins. Five main proteins were identified by mass spectrometry in all ACMs tested (Table 1). Many of these proteins are to be expected, including cystatin C, which has been observed in a previous proteomics experiment on astrocytic secretions [START_REF] Lafon-Cazal | Proteomic Analysis of Astrocytic Secretion in the Mouse[END_REF]. However, clusterin, which is a secreted sulphated glycoprotein was one of the major proteins found in midbrain and hippocampal ACM.

Interestingly, the MALDI-TOF/TOF-MS/MS database search software was not able to identify clusterin in the cortical ACM, suggesting that this protein, if present at all, is secreted at very low levels.

Clusterin increases neuronal differentiation from VM derived neurospheres.

To determine whether clusterin was responsible for increased neuronal differentiation immunodepletion studies were undertaken on midbrain ACM. In order to exclude any effect of the minimal media used for the conditioning, this was done in ACM (containing N2) as well as in ACM without N2 as was used for mass spectrometric analysis. Results show that there was a similar level of clusterin in normal media compared to media used for mass spectrometric analyses (Fig 1C ) with a rabbit serum control negative for clusterin (data not shown). We next wanted to determine if clusterin in our midbrain and hippocampal ACM was glycosylated. The two subunits of secreted clusterin can be resolved after treatment with endo-glycosidases. Fig 1D and Fig S1 show that secreted clusterin is highly glycosylated with more than 30% of its molecular weight corresponding to both O-linked and N-linked oligosaccharides. In order to determine the effect of clusterin on neuronal differentiation, neurospheres were differentiated in media, which had either had clusterin immunodepleted or still had it present. Results show that upon removal of clusterin (-sClu) there was a significant decrease in the number of TuJ1+ neurons and also pNF (Fig 1E). The rabbit serum control did not have any effect and neuronal differentiation was not significantly different from untreated ACM. This demonstrates that secreted clusterin is having a trophic effect on neuronal differentiation.

We next wanted to determine if the protein clusterin alone could increase neuronal number to a similar extent to midbrain ACM. Clusterin was added at a concentration of 10nM and found to significantly increase the number of TuJ1+ neurons (Fig 3A,D).

In order to determine if this was via an increase in cell survival or an increase in In order to determine if clusterin could increase neuronal differentiation from another brain region, cortically derived NPCs expanded under identical conditions as described for VM cultures, were treated with clusterin (5 or 10nM). Clusterin at 10nM (but not at 5nM) increased the number of TuJ1+ neurons (Fig 4A ,D), had no effect on cell proliferation (Fig 4B) but decreased TUNEL+ nuclei (Fig 4C ,D), similar to that reported above for midbrain NPCs. This demonstrates that the effects of clusterin are not region specific, although its secretion is.

Discussion

ACM derived from the hippocampus and midbrain increases neuronal differentiation from expanded human NPCs, as assessed by TuJ1 and SMI312. SMI312 identifies phosphorylated neurofilaments and has previously been shown to be axon specific [START_REF] Lubetzki | Even in culture, oligodendrocytes myelinate solely axons[END_REF]. Phosphorylation of neurofilaments during development is fundamental to maturation and stabilisation of the neuronal cytoskeleton, as well as an increase in axonal diameter [START_REF] Carden | To-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns[END_REF]. We show that hippocampal and midbrain ACM also increase the neurite length of phosphorylated neurofilament positive neurons indicating their role in neuronal maturation. Cortical ACM had no such effect. Most astrocytes are known to have similar morphological features, however they manifest functional differences in various CNS regions, probably due to the heterogeneity observed in their profile of membrane receptors, ion channels and gap junction coupling [START_REF] Hansson | Astrocytes in neurtransmission[END_REF]21,[START_REF] Lee | Astrocytes exhibit regional specificity in gap junction coupling[END_REF]. It is well known that astrocytes can produce soluble factors that influence function in the CNS; in fact their main functions were thought to be structuring the brain and supplying neurons with trophic support. However, it has recently been shown that astrocytes can promote neurogenesis from both adult neural and skin derived stem cells [START_REF] Song | Gage FH Astroglia induce neurogenesis from adult neural stem cell[END_REF][START_REF] Joannides | Efficient generation of neural precursors from adult human skin: astrocytes promote neurogenesis from skin-derived stem cells[END_REF] as well as mouse embryonic stem cells [START_REF] Nakayama | Astrocyte derived factors instruct differentiation of embryonic stem cells into neurons[END_REF].

Maldi-TOF analysis revealed a number of proteins were present in all ACMs tested. Cystatin C is a low molecular weight protein (13kDa), which is expressed in the brain of rat, monkey and man and is localised predominately in astrocytes but also in some neurons [START_REF] Yasuhura | Expression of cystatin C in rat, monkey and human brains[END_REF]. Interestingly, it has been purified from the conditioned medium of adult hippocampal neural stem cell cultures and has been shown to be required for the mitogenic activity of FGF2 [START_REF] Taupin | Gage FH FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor[END_REF]. In addition, its combined delivery with FGF2 in vivo to the adult dentate gyrus resulted in increased neurogenesis in this brain region [START_REF] Taupin | Gage FH FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor[END_REF]. Moreover, cystatin C has been shown to have a role in neuroprotection in vivo, for example, Olsson and colleagues demonstrated that focal ischaemia resulted in significantly greater brain infarcts in a cystatin C gene knockout mouse [START_REF] Olsson | Gene deletion of cystatin C aggravates brain damage following focal ischemia but mitigates the neuronal injury after global ischemia in the mouse[END_REF]. Furthermore, cystatin C has been shown to prevent the degeneration of rat substantia nigra dopamine neurons both in vitro and in vivo [START_REF] Xu | prevents degeneration of rat nigral dopaminergic neurons: in vitro and in vivo studies[END_REF]. Taken together these results demonstrate the importance of this protein in cell survival. Another 2 proteins found in ACM, α-2 macroglobulin and extracellular matrix 2 have been implicated in neurite outgrowth. α-2 macroglobulin is a major serum glycoprotein found in adult and fetal brain cells (29) including astrocytes [START_REF] Gebicke-Haerter | Alpha 2-macroglobulin synthesis in an astrocyte subpopulation[END_REF]. It has been shown to bind neurotrophic factors, such as NGF and NT3, which are secreted by astrocytes [START_REF] Lin | Protection of ischemic brain cells is dependent on astrocyte-derived growth factors and their receptors[END_REF]. We have previously shown that human NPC's express the trk C receptor [START_REF] Caldwell | Growth factors regulate the survival and fate of cells derived from human neurospheres[END_REF], so binding of α-2 macroglobulin to NT3 secreted by astrocytes may be playing a role in the neurite outgrowth seen with hippocampal and midbrain ACM. Additionally, extracellular matrix glycoprotein, falls into the family of neurite promoting factors, so this too may be playing a role in neurite outgrowth [START_REF] Lee | Rat alpha1-macroglobulin enhances nerve growth factorpromoted neurite outgrowth, TrkA phosphorylation, and gene expression of pheochromocytoma PC12 cells[END_REF].

The other major protein that was found in midbrain and hippocampal ACM is clusterin. This is a major secretory glycoprotein of 75-80kDa [START_REF] Blaschuk | Fritz IB Purification and characterization of a cellaggregating factor (clusterin), the major glycoprotein in ram rete testis fluid[END_REF], which to date has been implicated in several diverse physiological functions [START_REF] Rosenberg | Clusterin: physiologic and pathological considerations[END_REF][START_REF] Pucci | Neuroprotection: vEGF, IL-6 and clusterin: the dark side of the moon[END_REF]. It has been shown

to have greater expression in the brain than in other tissues [START_REF] Desilva | ApolipoproteinJ: structure and distribution[END_REF]. Its expression is highest in astrocytes, while its expression in neurons shows regional differences both in rat and human brain [START_REF] Pasinetti | Clusterin (SPG-2): a multifunctional glycoprotein with regional expression in astrocytes and neurons of the adult rat brain[END_REF]. Here we show that it has no effect on the proliferation of NPCs but it does promote their differentiation by sustaining ERK phosphorylation (for review see 39) and decreasing apoptotic nuclei. Interestingly, an increase in pERK has been associated with increased cell survival (for review see [START_REF] Nishida | The MAP Kinase cascade is essential for diverse signal transduction rathways[END_REF], so together our data on sustained pERK and decrease in TUNEL positive nuclei suggests clusterin is promoting both cell survival and differentiation. Interestingly, clusterin has previously been shown to have both pro and anti apoptotic potential. In the lipopolysaccharide model of glial activation, a process which occurs following injury, clusterin levels are increased 24 hours after its addition to astrocyte cultures [START_REF] Saura | Paul SM Microglial apolipoprotein E and astoglial apolipoprotein J expression in vitro: opposite effects of lipopolysaccharide[END_REF]. Indeed, several reports have shown that clusterin is a sensitive marker for different kinds of degeneration and neuronal injury [START_REF] May | Sulfated glycoprotein2: new relationship of this new multifunctional protein to Neurodegeneration[END_REF]. It is thought to play a neuroprotective role in Alzheimers disease (AD), because neurons expressing clusterin have enhanced resistance to neuronal death [START_REF] Giannakopoulos | Possible neuroprotective role of clusterin in Alzheimer's disease: a quantitative immunocytochemical study[END_REF]. Two recent large genome wide association studies demonstrated that clusterin is an important susceptibility gene for late onset AD [START_REF] Harold | Genome wide association study identifies variants at CLU and PICALM associated with Alzheimers disease[END_REF][START_REF] Lambert | Genome wide association study identifies variants at CLU and CR1 associated with Alzheimers disease[END_REF]. Indeed, clusterin has been found in the regions of the brain most affected in AD, the hippocampus and entorhinal cortex (for review see 46). However, clusterin is rarely found in neurons containing neurofibrillary tangles [START_REF] Giannakopoulos | Possible neuroprotective role of clusterin in Alzheimer's disease: a quantitative immunocytochemical study[END_REF]. In addition, the presence of ApoE4/4 allele significantly decreased the amount of clusterin in the frontal lobe in AD patients [START_REF] Harr | Brain expression of apolipoprotein E,J and A-I in Alzheimers disease[END_REF]. Together these observations suggest that the increased expression of clusterin in AD may represent a protective effect. In further support of its pro and antiapoptotic effects, studies using clu-/-mice, demonstrate that autoimmune myocardial damage is increased [START_REF] Mclaughlin | Apolipoprotein J/clusterin limits the severity of murine autoimmune myocarditis[END_REF] and clusterin has neuroprotective properties in permanent focal cerebral ischaemia [START_REF] Wehrli | Inhibition of postischemic brain injury by clusterin overexpression[END_REF][START_REF] Imlof | Sustained astrocytic clusterin expression improves remodeling after brain ischaemia[END_REF]. In contrast, in the same knockout strain the absence of clusterin reduces cell death in neonatal hypoxia-ischaemia via a caspase-3 independent brain injury pathway [START_REF] Han | Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia[END_REF], suggesting it functions to exacerbate neuronal damage under these circumstances. Interestingly, there are a number of other molecules such as nitric oxide (for review see 52) and HIF1a (for review see 53) that serve to promote or inhibit cell death depending on the cellular context and clusterin may fit into this same category.

In conclusion, we have shown that clusterin secreted from midbrain astrocytes, increases neurogenesis and neurite outgrowth from expanded human NPCs. This protein may have implications for repair in the CNS.

Legends

  Following washing, Pansorbin A beads were incubated in either anti rabbit clusterin (0.1mg) or normal rabbit serum (Invitrogen) overnight at 4 o C. Following x3 PBS washes; beads with absorbed IgGs were incubated with 5 ml of midbrain ACM for 6 hours at 4 o C that had been collected in N2 containing medium or medium used for Mass Spec analysis (without N2). Beads were pelleted by centrifugation. ACM was collected for neuronal differentiation and beads were boiled for 5 mins in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EGTA) containing a cocktail of protease inhibitors (Complete, Mini, Roche) and centrifugation at 14,000 rpm for 20 min at 4 0 C. Deglycosylation of conditioned media 18 ul of ACM from Midbrain and Hippocampus were incubated for 10 min at 100ºC with 2 μl of denaturing buffer (NEB). Denatured ACMs were treated with 500 U of peptide:Nglycosidase F (PNGase F, NEB) to remove N-linked oligosaccharides; 40000 U of endo-α-Nacetylgalactosaminidase (O-glycosidase, NEB) and 50 U of neuramidase (NEB) to eliminate O-linked oligosaccharides. Reactions were carried out in 1% NP40 and 1x G7 reaction buffer at 37ºC overnight. Deglycosylated extracts were run in 12% SDS-Tris-Glycine gels and probed for clusterin.

  15000 cells were plated on poly-D-lysine coated 96-well plates. 24h later cells were stimulated with 10nM clusterin (Prospecbio) for between 5 min and 6 hours. Cells were fixed with 4% PFA and stained with anti-p44/42 MAP Kinase and anti-phospho p44/42 MAP Kinase overnight. Appropriate secondary antibodies were used (Suppl T2). Image acquisition and analysis was performed on an IN Cell Analyzer workstation (GE Healthcare) using a Multi Target Analysis algorithm to quantify fluorescence intensity in nuclear and cytoplasmic compartments (in total and ppERK1/2 image channels).

Fig

  Fig 1A), while hippocampal ACM had an effect only after a month (P<0.05).

  proliferation, TUNEL staining and BrdU analysis were performed. Results show that 10nM clusterin decreased the number of apoptotic nuclei compared to untreated cells (Fig 3B, D) but did not significantly affect cell proliferation (Fig 3C, suppl Fig 2). Interestingly, 10nM clusterin reduced the number of TUNEL+ nuclei to a similar extent to midbrain ACM suggesting this protein is present at a similar concentation in midbrain ACM. Furthermore 10nM clusterin caused a sustained increase in ERK phosphorylation indicating its potential to regulate cellular differentiation and survival (Fig 3E, F).

Figure 1 .

 1 Figure 1. ACM increases neuronal differentiation from VM derived NSCs. A,

Table 1 .

 1 Proteins identified in the conditioned medium of cultured astrocytes from various brain regions. The numbers of peptides observed by MALDI-TOF/TOF tandem mass spectrometry and the percentage of amino acid sequence covered for each protein is indicated.

Figure 2 .Figure 3 .

 23 Figure 2. Clusterin or regional ACM does not influence GFAP positive cells

Figure 4 .

 4 Figure 4. Clusterin increases neuronal differentiation from cortical derived

Table 1

 1

			Mw	Midbrain	Hippocampus		Cortex
		Number	(kDa)	Peptides Coverage Peptides Coverage Peptides Coverage
	α-2-macrogobulin	6978425	164	10	8.7%	6	6%	3	3%
	clusterin	17985951	51	4	11%	4	11%	-	-
	cystatin C	226712	13	3	39%	3	30%	3	39%
	extracellular matrix protein 2	6978789	70.5	2	6%	-	-	4	10%
	vimentin	1353212	53.7	2	6%	8	22%	11	27%

Acknowledgements.

This work was funded by an MRC Studentship (SAS), University of Bristol Studentship (OC-L) and the Royal Society and Parkinsons Disease Society (MAC). RAB and PT receive support from the NIHR grant of a Biomedical research centre to the University of Cambridge and Addenbrooke's Hospital, and the MRC for fetal tissue collection. CMK and AER receive support from the MRC (UK) and Wales Office of Research and Development (WORD) for human fetal tissue collection. The INCELL analyser was funded by the Wellcome Trust Equipment Grant (078407) to Craig A. McArdle, University of Bristol.

Conflict of Interest statement.

The authors declare no conflict of Interest.


Hernandez Camilo

Bernard Yves

Adel Razek

A Global Assessment Of Piezoelectric Actuated Micro-pumps

Keywords: Piezoelectric, micro-pump, peristaltic, nozzle/diffuser valve, cantilever valve, ball valve, bridge valve

This article provides an extensive outlook of different types of piezoelectric actuated micro-pumps published in the literature recently. We start by reminding the reader about the conventional operating parameters used to quantify the capabilities of these devices. After this introductory stage, we describe and classify the most prominent micro-pump's geometries found with piezoelectric actuation. At this point we identify the parameters given previously for each pump in order to establish a subsequent discussion in which the trends of different families are compared. Specific attention is given to the particularities of each case namely: flow-rates and back-pressures

Introduction

Micro-pump's applications are found in large variety of technology and science domains such as biology, medicine, electronics and space air crafting. They are used in micro total analysis systems (µTAS), portable DNA testing devices, electronic refrigeration systems and portable medication delivery appliances mainly for insulin distribution among others. As a rule, micro-pumps are designed and manufactured to meet a set of requirements on parameters whose importance may vary with the application; therefore it is very common to find significant differences from one to another. For example, in the field of drug delivery systems (DDS), it is known that the efficiency of some drugs is directly related to the way it is distributed [START_REF] Shawgo | BioMEMS for drug delivery[END_REF].

In fact, some medications are more effective when they are injected into the body in definite concentrations. For this reason, micro-pumps designed for drug delivering purposes are required to pump particular quantities of fluid with high controllability [START_REF] Teymoori | Design and simulation of a novel electrostatic peristaltic micromachined pump for drug delivery applications[END_REF], taking into account some other important particularities such as reduced size, low power consumption and biocompatibility [START_REF] Nisar | MEMS-based micropumps in drug delivery and biomedical applications[END_REF]. Other applications may demand different features from micro-pumps. In the case of electronic cooling systems for example, the key points for designers are high flow rates and large pressure heads [START_REF] Iverson | Micropumping Technologies for Electronics Cooling[END_REF]. These qualities make the pump appropriated to be incorporated in the circuitry of modern electronic devices.

A significant selection of actuation principles is suitable to solve problems in domains where micropumps are normally used. Magnetohydrodynamic [START_REF] Lemoff | An AC magnetohydrodynamic micropump[END_REF], electroosmotic [START_REF] Wang | Electroosmotic pumps for microflow analysis[END_REF], electrohydrodynamic [START_REF] Darabi | Development of an electrohydrodynamic injection micropump and its potential application in pumping fluids in cryogenic cooling systems[END_REF], pneumatic [START_REF] Takao | A MEMS microvalve with PDMS diaphragm and two-chamber configuration of thermo-pneumatic actuator for integrated blood test system on silicon[END_REF], [START_REF] Galas | Characterization of pneumatically activated microvalves by measuring electrical conductance[END_REF], electrostatic [START_REF] Luharuka | Simulated and experimental dynamic response characterization of an electromagnetic microvalve[END_REF], [START_REF] Bosch | A silicon microvalve with combined electromagnetic/electrostatic actuation[END_REF], piezoelectric are some examples of the drivers most frequently employed in the design of these devices. A complete description of several types of micro-pumps can be found in [START_REF] Laser | A Review of Micropumps[END_REF]. However, piezoelectric actuation presents the particularity of being very likely to meet many of the requirements of a vast range of domains. Advantageous properties such as high integration capacity in micro-systems, low power consumption, high efficiency and high speed response make of piezoelectricity a very good choice for designers when it comes to design micro-pumps. In comparison to devices with different actuation principles, piezoelectric micro-pumps can be designed to work at very high frequencies which translates to increasing flow-rates. This feature is in opposition to pneumatic or shape memory alloy actuation for example. In addition, piezoelectric ceramics present large force generation capacities and the ability of being synthesized and shaped at very small scale permitting their integration in micro-devices. These two last characteristics combined give the possibility to create powerful and small devices. Feature, more difficult to obtain with other type of actuators such as electromagnetic, thermopneumatic and magnetohydrodynamic. In spite of these remarkable advantages, the main drawback of piezoelectric transducers is the low magnitudes of deformation and therefore of displacement, compelling the frequent use of amplifiers.

This article provides an outlook of the most recent piezoelectric micro-pumps found in the literature. The devices are presented and classified according to the type of motion applied to the fluid in order to create flow. For every geometry exposed in this paper, an analysis including their description, the elucidation of their working principle and the identification of their strengths and weaknesses with respect to the parameters used to quantify their performances is effectuated. Mainly two actuating motions stand out in the family of piezoelectric micro-pumps which will give the guidelines to classify the different architectures. On one side, there is the reciprocating displacement or piston-like motion. On the other side, there is the peristaltic movement. Within the latter category, we find continuous and discrete types. They are differentiated essentially by the interruptibility of the forces applied on the fluid. Inside the reciprocating group on the other hand, the difference is made basically by the type of valves used to rectify the flow. Here, we find nozzle-diffuser valves, check valves and a variety of actuated valves. Finally, a discussion is proposed. At this point, we mainly draw the attention to table 1 which summarizes the parameters of a variety of micro-pumps belonging to the categories presented before. At the beginning of this section, orders of magnitude of these parameters are presented and commented. This stage allows to constitute increasing or decreasing tendencies of certain variables according to the family of pump considered. At the same time, a comparison between different categories is carried out. Manifestly, this is done by keeping in mind the diversity of constraints and considerations made at the moment of designing and manufacturing.

Operating parameters

The very basic purpose of a micro pump is to displace a volume of fluid or gas from one place to another. For this reason, the parameters used to quantify the performance of such a device are related to the rate of fluid delivered per unit time and the maximum pressure under which the fluid can be moved.

When it comes to micro-pumps, the first concept that emerges is the maximum flow rate (Q max ). It quantifies the volume of liquid displaced per unit time when no pressure is exerted at the outlet. However, this case never happens in operating circumstances since there is always some pressure acting in opposition to the flow. When it happens, the flow rate starts diminishing with the pressure exerted. Due to this reason, it is very important to characterize a micro-pump according to the maximum pressure it can work against, which is normally known as the maximum backpressure (p). This parameter can be more formally defined as the pressure developed at the outlet of the pump due to the increment in flow velocity generated by the pump [START_REF] Ho | Micro/Nano Technology Systems for Biomedical Applications: Microfluidics, Optics, and Surface Chemistry[END_REF].

At maximum backpressure, the flow rate becomes zero. Actually, Q max and p give two points of a curve that provides an operation point for the pump. For a given pressure there is a unique value of flow rate and vice versa. Another conventional parameter used to measure the performance of a micro-pump is the pump head (h). It corresponds to the work done on a unit weight of liquid passing from the inlet to the outlet. In its simplified form [START_REF] White | Fluid Mechanics[END_REF], the pump head is calculated as the difference of pressure between the inlet and outlet divided by the specific weight (ρg):

g P P h in out ρ - =
Pump power (P pw ) is another parameter frequently used as well; it is defined as the power delivered to the fluid by the device, its value is calculated as the product of the backpressure and the flow rate. Pump power leads to the concept of pump efficiency (η), which is basically the ratio between the pump power and the power required to drive the actuator. The difference between these two is due to three type of looses according to [START_REF] Nisar | MEMS-based micropumps in drug delivery and biomedical applications[END_REF]. Fluid leakage losses which have direct impact on volumetric efficiency, frictional losses related to mechanical efficiency and losses due to imperfections in manufacturing associated to hydraulic efficiency. Therefore the total efficiency can be obtained from these three. Authors usually report the operating voltage and the frequency at which the actuator is submitted, since superior discharge volumes and flow rates are achieved when resonant modes of the piezoelectric structure are employed at certain levels of voltage. Normally, piezoelectric drivers demand high voltages (60V -1000V) due to the high electric fields required to deform the actuator considerably. Operating frequencies in the same way may vary significantly according to the operating principle and design. Additional to these parameters, others like package size, power consumption and maximum force exerted by the actuator are commonly reported on literature according to their significance on the design which is highly related to the application domain and the pump's configuration itself. At this point, it is worthwhile to remind the reader about the importance of the size of the pumping chamber since it plays a role in the order of magnitude of the pump parameters. Specifically, in cases where precision is demanded, the size of the pumping chambers tend to reduce in order to assure small pumped volumes. [START_REF] Woias | Micropumps-summarizing the first two decades, Microfluidics and BioMEMS[END_REF] also indicates the influence of a varying backpressure in the dosing accuracy of a micropump concluding that the pressure-dependent flow curve should be sufficiently flat to ensure an accurate dosing at varying outlet overpressure.

Reciprocating micro-pumps

The operation of this type of micro-pump is based on the change in volume of a two openings-reservoir containing a fluid. This volume change is better known as stroke volume (ΔV). In contrast to the stroke volume, there is the dead volume which corresponds to the quantity of liquid that remains in the pump when the chamber volume is minimal over a pumping cycle. The ratio of stroke volume and dead volume constitute a parameter known as compression ratio which is quite related to the pump's performance. A schema of this architecture is shown in figure 1.

Inlet Outlet Pumping Chamber Pump Diaphragm Fig. 1. Reciprocating Micro-pump
The rectification of the flow is normally effectuated by valves that according to their operating principle and geometry increase or reduce the complexity, power consumption and performance of the pump. For this reason, it is very important to subdivide this category under the criterion of the type of valves used. Three main types of valves are noticeable in reciprocating geometries. In a first place, there are the nozzle-diffuser valves which utilize exclusively particular shapes and geometries to direct the flow. Consequently, no moving parts are present. A second group is determined by the check valves, which posses moving parts and control the flow according to its direction. They privilege streams traveling in certain directions. Finally, there are the actuated valves which utilize external power sources to generate direct mechanical action on the fluid. When a reciprocating micropump is driven by piezoelectrics, it becomes very interesting to work at frequencies close to the resonance of the actuator in order to obtain higher displacements and consequently higher stroke volumes. Here, it is important to consider the viscosity of the fluid. [START_REF] Wang | Simulations and analysis of a piezoelectric micropump[END_REF] and [START_REF] Cui | Study on a piezoelectric micropump for the controlled drug delivery system[END_REF] propose a modal analysis of the piezoelectric transducer to establish the frequency at which the maximum stroke volume is obtained.

The core of a reciprocating micro-pump is undoubtedly the pumping chamber along with the pump membrane or pump diaphragm. The device's performance is directly related to the physic characteristics of these two latter. [START_REF] Nguyen | Fundamentals and Applications of Microfluidics[END_REF] illustrates the calculation of stroke volume according to different deflections patterns of the pump diaphragm for a cylindrical chamber. These computations will lead to the estimation of most of the parameters of this type of pump. Some authors like [START_REF] Mu | Optimisation design of a piezoelectric micropump[END_REF], design their devices to work at the resonant frequency of the pump diaphragm in order to improve the flow rate and the efficiency. At low frequencies, it can be assumed a linear relation between flow rate and pumping frequency (f), assuming a fluid displacement in one direction only:

f V Q ⋅ Δ =

Reciprocating micro-pumps with nozzle/diffuser valves.

The idea behind this geometry is to exploit the direction dependent behavior of tapered channels to rectify the flow. Two principal advantages stand out in micro-pumps having this type of valves. Firstly, nozzle/diffuser configuration avoids the clogging problem of moving valves. Secondly, structures become simpler reducing in this manner manufacturing complexity and costs. The main drawback of this type of micro-pumps is its low efficiency since there is a considerable amount of fluid re-entering the pumping chamber in a single pumping cycle. The performance of the rectification capability of a nozzle/diffuser configuration is measured by the ratio of the pressure loss at the inlet and the outlet (η nd ). This value depends on the geometry of the nozzle/diffuser [START_REF] Reece | Progress in Smart Materials and Structures[END_REF] (Diameter, length and tapper angle) and on the fluid characteristics (Kinematic viscosity, Reynolds number and flow velocity). Its magnitude is given by: With Re n , Re d , D 0 and D 1 standing for the Reynold's number of the nozzle, the Reynold's number of the diffuser, the diameter of nozzle and the diameter of diffuser respectively. For nozzle/diffuser geometries, the flow rate can be calculated as:

⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ =
1 1 2 + - ⋅ Δ ⋅ = nd nd f V Q η η For consideration 3/10
For consideration

4/10

Different applications can be found for nozzle/diffuser pumps. [START_REF] Tt | Performance evaluation of a valveless micropump driven by a ring-type piezoelectric actuator[END_REF] exposes a micro-pump designed to supply ethanol to a fuel cell with a flow rate requirement is about 0.31 ml/minute if using a methanol water solution as fluid. [START_REF] Ederer | Piezoelectrically driven micropump for ondemand fuel-drop generation in an automobile heater with continuously adjustable power output[END_REF] reveals a device conceived for fuel dosing in automobile heaters with the particularity of having a micro filtering system embedded at the inlet. [START_REF] Cui | Study on a piezoelectric micropump for the controlled drug delivery system[END_REF] moves to the field of medicine and proposes a micro-pump applied on an invitro injection system for diabetics. The author highlights the possibility of pumping gases and liquids, high reliability and low sensitivity of drug particles due to the lack of a moving valve.

Many approaches have been reported to improve the performance of this type of pumps. [START_REF] Andersson | A valveless diffuser micropump for microfluidic analytical systems[END_REF] presents a reciprocating micro-pump with high compression ratio composed of a shallow pumping chamber made from Silicon, a thin pumping diaphragm and a set of deep diffusers that according to the author improves the efficiency of the device. [START_REF] Cui | Simulation and optimization of a piezoelectric micropump for medical applications[END_REF] mentions similar statements about better performances of reciprocating micro-pumps when the thickness of the diaphragm is reduced. Actually, this article discusses the impact on flow rate when parameters like diffuser's geometry, excitation voltage and frequency are modified. All parameters involved in the optimization process are set in order to maximize the performance of the final architecture. An original improvement to the nozzle/diffuser geometry is proposed by [START_REF] Izzo | Modeling and experimental validation of a piezoelectric micropump with novel no-moving-part valves[END_REF]. Here, the classic tapered shape of the channels are modified by adding vortex areas along the fluidic pattern, which is, a sequence of rounded and sharp bifurcations added along the nozzle/diffuser path. The author claims to reduce the tendency of the flow to re-enter the pumping chamber and makes a comparison with a classical nozzle/diffuser geometry reporting an increment of efficiency of up to 2.9 times at maximum flow rate conditions. [START_REF] Gerlach | Working principle and performance of the dynamic micropump[END_REF] suggests a change in the geometry of the cross section of the nozzle/diffuser and studies its effect on the pump's performance.

Other authors propose deep changes to the classic structure of this type of pump. [START_REF] Yih-Lin | Manufacture of three-dimensional valveless micropump[END_REF] unveils a three dimensional architecture where the nozzle and the diffuser are not placed in the same plane claiming to be a suitable approach to generate pre-assembled valveless micro-pump structure.

Reciprocating micro-pumps with check valves

Check valves are those whose operation is defined by the direction of the flow. In fact, when they are embedded in a reciprocating structure, the forces created by changes in pressure inside the pumping chamber during a pumping cycle move a two-position mobile which allows or stops the fluid's circulation.

According to piezoelectric micro-pump's literature so far, the mobile can take mainly three forms as shown in figures (2a), (2b) and (2d): First (figure 2a), there is the ball-valve type where the mobile is a ball which blocks an opening by the action of the fluid's pressure exerted on it. Similarly, when the flow goes in the opposite direction, the pressure of the fluid removes the ball away from the opening allowing liquid to pass. An example of this geometry is given by [START_REF] Accoto | Modelling of micropumps using unimorph piezoelectric actuator and ball valves[END_REF]. The paper presents micro-pump made with a stereolithographic technique composed of steel balls of 1,2 mm diameter and a valve stopper of 0,5 mm diameter. Additionally, an approach aimed to predict the performance of a ball valve micro-pump versus the working frequency according to its geometrical and mechanical characteristics is discussed.

In the second place (figure 2b), there is the flap-valve. The operating principle is exactly the same as that of ball-valves. However in this geometry, the mobile is replaced by a cantilever beam fixed at one of its tips. The beam bends upwards/downwards allowing circulation or remains straight blocking it. All according to the flow direction. The design and study of this type of geometry is accomplished by considering the dynamic behavior of the device. In fact, many authors rely on the great displacements obtained on the cantilever beam when it is driven at its resonant frequency. [START_REF] Ma | Development and application of a diaphragm micro-pump with piezoelectric device[END_REF] for example, presents a micro-pump with flap valves and an innovative diaphragm geometry. The author models this latter with a classic mass-damper-spring system and makes use of the harmonic resonance of the working fluid along with the system components to drive the whole at a frequency that optimizes the device's performance. As conclusion of the work, it is claimed that better results were obtained by using narrow valves. In a similar way, [START_REF] Kan | Design and test of a high-performance piezoelectric micropump for drug delivery[END_REF] makes a discussion in his work about the influence of the resonant frequencies of the valves and the actuator in the performance of the micro-pump. Actually, the author draws attention to the maximum flap valves efficiency observed when they work at their resonant frequency and the impact on the pump's flow rate. The author claims as well a peak in the device's performance when it is driven at the resonant frequency of the piezoelectric actuator, suggesting multiple optimal frequencies at which the pump can work.

Other authors rely exclusively on the resonant frequency of the actuator. [START_REF] Park | Resonantly driven piezoelectric micropump-fabrication of a micropump having high power density[END_REF] for example, increases the inertial mass of the piezoelectric transducer in order to adapt its resonant frequency for a pumping application. As final example of this type of micropump, there is that presented by [START_REF] Feng | Piezoelectrically Actuated Dome-Shaped-Diaphragm Micropump[END_REF]. The core of his design is a domed shaped diaphragm which is supposed to release extra pressure due to the residual stress stored in the domed shape. Finally (figure 2c), there are the bridge-valves that work very closely to the flap ones. Here, the opening is blocked by a plate attached to the structure of the pumping chamber by four micro-springs located in every corner. Again, if the flows goes in the right direction, the plate goes up allowing circulation through the gap between it and the valve stopper.

Otherwise, the flow is blocked by the plate itself. This type of valves present a more complicated manufacturing process, however they are claimed to support great values of pressure. In fact, [START_REF] Li | Development of large flow rate, robust, passive micro check valves for compact piezoelectrically actuated pumps[END_REF] discusses the tendency of flap valves to fail under extreme high pressures and the convenience of having four contact points with the pump structure instead of one which is the case of the flap-valve. The author presents a micro-pump actuated by a piezoelectric stack supporting 350 PSI at 10 kHz. [START_REF] Nguyen | A fully polymeric micropump with piezoelectric actuator[END_REF] proposes in a similar way a valve composed of a disc over a compliant orthoplanar string with four folded arms made from SU-8 which is an epoxy-based negative photo-resist. This material has the advantage of high flexibility so the micro-strings require less fluid pressure to work. The pump unveiled in this article operates at 1 mL/min at a pressure of 200 mm of water.

In general, reciprocating pumps with check valves have better performance than those with nozzle/diffuser. They are known for having high compression ratios and high pump pressures [START_REF] Richter | Robust Design of Gas and Liquid Micropumps[END_REF].

Reciprocating micro-pumps with actuated valves

In addition to the compulsory need for external sources of energies to create motion on a fluid in mechanical micro-pumps, this category uses also actuating forces to rectify the flow. Characteristic aspects of this type are: the increased complexity of the device due to the electronics embedded to control the opening and closing sequences of the valves.

There is also the extremely high pressures stood by the pump and consequently high efficiency values which is a direct consequence of the fact that the valve blocking is done by external actuators. Actuated valves are employed normally for specific applications. For example, [START_REF] Doll | A high performance bidirectional micropump for a novel artificial sphincter system[END_REF] exhibits a pump conceived for a medical application. More specifically, it is applied in a novel sphincter system composed by a cuff system which inflates or deinflates with a biological compatible fluid replacing in this way a weaken sphincter muscle. The micropump used in this application moves fluid inside the cuff system. Therefore, high flow-rates to allow rapid artificial muscle contractions and high pressures (around 30 kPa) are required. The valves and the pumping chamber of this device are actuated by piezoelectric patches. The valves have also a nozzle/diffuser geometry that increases even more the performance of the pump. The author highlights features such as biocompatibility and high pressure (60 kPa at 1,8 mL/min).

Peristaltic micro-pumps

The word peristaltic comes from the Greek peristaltikos which relates the movement caused by propagating muscular contractions that take place in the intestines. The same basis is then applied to create fluid flow. This principle is often found in piezoelectric actuated micro-pumps either under the form of multiple pumping chambers connected in series actuated harmonically by independent sources, or, under the form of a flexible channel whose deformation follows a mechanical travelling wave motion. These arrays can either be linear or circular.

There are two types of structures of peristaltic micro/pumps. The first corresponds to a discrete or discontinuous geometry since the excitation is localized on several independent points (figure 3a). The second on the contrary, depicts a continuous architecture in which the motion is applied over the entire structure (figure 3b). A remarkable feature of this type of micro-pump is the absence of valves that makes of this category a

For consideration 5/10

For consideration 6/10 good choice when manufacturing simplicity is an issue.

Discrete peristaltic micro-pump

This geometry is basically an array of multiple reciprocating pumping chambers (at least three) connected in series and actuated sequentially to move the fluid in one direction or other. The principal advantage of this architecture is the possibility of reversing the pumping direction. However, it suffers from the same inconvenient of the nozzle/diffuser reciprocating micro-pump, which is low efficiency caused by fluid re-entering the pumping chambers. Additionally, there is an increment in power consumption and the necessity of having a control system to generate the pumping sequence. The stroke volume is calculated following the same approach as for reciprocating pumps consequently the frequency at which the device is actuated is a critical issue for design. [START_REF] Lee | Bidirectional pumping properties of a peristaltic piezoelectric micropump with simple design and chemical resistance[END_REF] proposes a 12 µL/min micropump with a maximum back pressure of 3 kPa composed of three pumping chambers applied to the control of biosamples. The author discusses the effect of the membrane resonant frequency on the device's performance and utilizes it as a mean to obtain the optimal pumping sequence speed. Similarly, [START_REF] Jang | A stand-alone peristaltic micropump based on piezoelectric actuation[END_REF] and [START_REF] Jang | Peristaltic micropump system with piezoelectric actuators[END_REF] suggest a relationship between the actuator activation sequence and the resonant frequency of the diaphragm. The author reports a change in resonant frequencies of diaphragm when the sequence varies. Subsequently, the flow rate is affected. Another very interesting geometry that can be inscribed in this family of pumps is proposed by [START_REF] Kanno | Development and characterization of a traveling wave micropump driven by piezoelectric actuators[END_REF].

Here, a mechanical traveling wave motion is created by an array of piezoelectric transducers placed under a flexible channel actuated in such a manner that they deform the walls of this latter creating in this way a peristaltic motion. The maximum flow rate generated by this micro-pump is 1 µL/min.

Linear peristaltic micro-pump

In this configuration, the motion of fluid is generated by the action of a mechanical travelling wave deflecting the walls of the pumping chamber. The principal characteristic of this type of micro-pump is the high frequencies of operation which are usually greater than 20 kHz. Normally the geometries belonging to this category are simpler and do not require additional controllers to generate sequences. The drawback lies on the difficulty to obtain a mechanical travelling wave in a finite elastic medium. [START_REF] Cohen | [END_REF] has registered a patent of a micro-pump following this principle claiming as advantages the simplicity, the absence of moving parts and a great capacity of integration.

Discussion

The table 1 exposes the principal characteristics of several micro-pumps published over the last two decades. A variety of characteristics, operating conditions, material and performances can be observed.

At this stage, it is possible to make some inferences about the order of magnitude of the most significant parameters of micro-pumps.

For example, when it comes to flow-rate, the most diversified ranges are found in pumps having nozzlediffuser and cantilever valves. Their magnitudes go from some µL/min to some thousands of µL/min. This fact makes of them the most heterogeneous categories. Micro-pumps having actuated valves and those peristaltic posses more uniform values of flow-rates. They lay around tens of µL/min and some thousands of µL/min respectively. When magnitudes of back-pressure are considered, nozzle-diffuser are once again the most varied family.

Here, their values range between few Pa and thousands of Pa. Peristaltic pumps on their own side work in the range of hundreds to thousands of Pa. Finally, for pumps having cantilever and actuated valves, the back-pressure range is about several thousands of Pa. The orders of magnitude of micro-pump's package size were determined from examples published in literature likewise. Sizes of devices using nozzlediffusers lay around the same range, namely, few mm 3 . To site some examples, there are those from [START_REF] Ederer | Piezoelectrically driven micropump for ondemand fuel-drop generation in an automobile heater with continuously adjustable power output[END_REF], [START_REF] Izzo | Modeling and experimental validation of a piezoelectric micropump with novel no-moving-part valves[END_REF] and [START_REF] Yih-Lin | Manufacture of three-dimensional valveless micropump[END_REF]. Peristaltic pumps exist within a range between some tens of mm 3 [START_REF] Kanno | Development and characterization of a traveling wave micropump driven by piezoelectric actuators[END_REF] and few tens of cm 3 [START_REF] Lee | Bidirectional pumping properties of a peristaltic piezoelectric micropump with simple design and chemical resistance[END_REF]. Last, micro-pumps having actuated valves and cantilever valves show similar size characteristics. The average order of magnitude is located around few mm 3 as it is exemplified by [START_REF] Park | Resonantly driven piezoelectric micropump-fabrication of a micropump having high power density[END_REF] or [START_REF] Doll | A high performance bidirectional micropump for a novel artificial sphincter system[END_REF].

Voltage levels on the contrary are very uniform in general, they vary around one hundred volts. However, exceptionally, they can take small values as in the pump reported by [START_REF] Doll | A high performance bidirectional micropump for a novel artificial sphincter system[END_REF].

We remind the reader at this point that there is no indication that a new or existing pump could not take values beyond the limits presented here. Since, these were based on few available examples that belong to a large population. When the magnitudes presented in table 1 are observed more narrowly, it is noticeable that no huge differences between different categories appear. However, certain generalizations can be made. Manifestly, this generalizations are not meant to work as rules since clever designs, bigger pumping chambers and other innovations can improve the performances of certain geometries over others. For example, the highest back-pressures are detected in reciprocating micro-pumps having actuated valves and cantilever valves which is coherent with the description given previously. The maximum value found on the table regarding back-pressure is provided by the pump published by [START_REF] Park | Resonantly driven piezoelectric micropump-fabrication of a micropump having high power density[END_REF] which belongs to the cantilever type-valve. This device was designed to work at the resonant frequency of a mass-piezostack system which increments dramatically the inertial mass and the pump power supplied to the fluid. It is also interesting to highlight the high values of flow rate in structures conceived to avoid fluid reentering the pumping chamber. Those are reciprocating pumps with actuated valves, bridges and cantilevers.

Additionally, standing out among all the examples given in this article, there is the pump reported by [START_REF] Tt | Performance evaluation of a valveless micropump driven by a ring-type piezoelectric actuator[END_REF] which despite belonging to a geometry that by nature is not likely to have high flow rates, presents the highest value. Two factors might explain this situation. One one side, there is the fact of driving the whole loaded device at its resonant frequency (fluid included). On the other side, there is the relative large volume of the pumping chamber which allows important amounts of fluid displaced per pumping cycle. Another remarkable example is the pump presented by [START_REF] Izzo | Modeling and experimental validation of a piezoelectric micropump with novel no-moving-part valves[END_REF] who reports a 10900 Pa nozzle/diffuser micro-pump. This feature can be explained by the vortex areas added to the classic nozzle/diffuser path increasing in this way the ability of the device to stand higher pressures . As for the rest of the nozzle/diffuser micro-pumps, the expected behaviour is evidenced namely, modest flow rates and back-pressure values.

As last comment, there is a trend among designers to work with Silicon due to the vast diffusion of this material in the manufacturing of micro-systems.

Conclusions

We have presented an article aimed to give an understanding and a global view of the different and most popular types of piezoelectric micro-pumps reported so far. Starting with the basic concepts about the characterization of these devices, we have made a tour over a variety of families highlighting every time the most notorious features and some designing principles.

Several examples were presented through the paper as well as a brief discussion over the generalizations and tendencies of every architecture.

Regarding piezoelectric actuated micro-pumps literature, it was rather clear that the reciprocating devices were the most frequently reported. In fact, most of the examples given in this article belong to this category. Advantages like manufacturing ease, intuitive functioning and adaptability to multiple applications make of this type the most evident choice for designers when a micro-pump is needed. The tendency in general is to go for the simplest structure, evidence of that is the high number of publications regarding reciprocating piezoelectric pumps having nozzle/diffuser valves. At this point it is worth to say that every micro-pump was designed for a purpose and there is no superiority from one to another. Every family has defined features that are more or less adaptable to certain utilizations and operating conditions.

Table 1. Comparison of piezoelectric actuated micro-pumps

Fig. 2 .

 2 Fig. 2. Check valves. (a) Ball, (b) Flap, (c) Bridge

Fig. 3 .

 3 Fig. 3. Peristaltic micro-pumps. (a) Discrete geometry, (b) Continuous geometry.

For consideration

For consideration 8/10

For consideration 9/10


James H Rice

Graeme A Hill

Stephen R Meech

Paulina Kuo

Konstantin Vodopyanov

Michael Reading

Sub-wavelength surface IR imaging of soft-condensed matter

Outlined here is a technique for sub-wavelength infrared surface imaging performed using a phase matched optical parametric oscillator laser and an atomic force microscope as the detection mechanism. The technique uses a novel surface excitation illumination approach to perform simultaneously chemical mapping and AFM topography imaging with an image resolution of 200 nm. This method was demonstrated by imaging polystyrene micro-structures.

Introduction

Infrared spectroscopy (IR spectroscopy) is a powerful and well developed method to characterise the chemical composition of materials. IR spectroscopy utilises infrared radiation commonly from a black body emitting source to tune across vibrational absorption frequencies [START_REF] Stuart | Infrared spectroscopy: fundamentals and applications[END_REF]. IR is frequently applied to characterise molecular species by utilising the characteristic vibrational modes (eigenmodes) that each molecular species possess.

Microscopy based methods have made great progress in developing techniques that can image on the nanoscale, these approaches can be divided into two main classes, near-field and far-field techniques. IR Imaging using far-field methods is limited by diffraction to length scales of the order of 1-5 micrometers for IR microscopy [START_REF] Betzig | Breaking the Diffraction Barrier: Optical Microscopy on a Nanometric Scale[END_REF]. It is noted that fluorescence based imaging has successfully over come the diffraction limit in the far-field to enable an image resolution as good as tens of nanometres to be archived [START_REF] Rice | Fluorescence microscopy beyond the diffraction limit: Fluorescence microscopy with super-resolution[END_REF], however to date no progress has been reported for far-field IR based imaging.

Scanning probe techniques such as Atomic force Microscopy (AFM) enable topographic imaging of surfaces with very high spatial resolution, i.e. < 10 nm [START_REF] Albrecht | Atomic resolution with the atomic force microscope on conductors and nonconductors[END_REF].

Such an approach to imaging is a well developed near-field method. Combining IR spectroscopy with AFM potentially allows for a method that can chemically map materials with nanometre spatial resolution. Such an approach based on combining AFM and IR spectroscopy has been developed [START_REF] Hammiche | Photothermal FT-IR spectroscopy: A step towards FT-IR microscopy at a resolution better than the diffraction limit[END_REF][START_REF] Hammiche | Mid-infrared microspectroscopy of difficult samples using near-field photothermal microspectroscopy[END_REF][START_REF] Hammiche | Progress in near-field photothermal infra-red microspectroscopy[END_REF][START_REF] Reading | Micro-thermal analysis of polymers: Current capabilities and future prospects[END_REF][START_REF] Dazzi | Subwavelength infrared spectromicroscopy using an AFM as a local absorption sensor[END_REF][START_REF] Dazzi | Analysis of nano-chemical mapping performed by an AFM-based ("AFMIR") acousto-optic technique[END_REF][START_REF] Houel | Ultraweak-Absorption Microscopy of a Single Semiconductor Quantum Dot in the Midinfrared Range[END_REF][START_REF] Mayet | Sub-100 nm IR spectromicroscopy of living cells[END_REF][START_REF] Hill | nano-Infrared surface imaging using an OPO and an AFM[END_REF][START_REF] Aizpurua | Substrate-enhanced infrared near-field spectroscopy[END_REF][START_REF] Cvitkovic | Material-specific infrared recognition of single sub-10nm particles by substrate-enhanced scattering-type near-field microscopy[END_REF][START_REF] Brehm | Infrared spectroscopic mapping of single nanoparticles and viruses at nanoscale resolution[END_REF][START_REF] Formanek | Analysis of the measured signals in apertureless near-field optical microscopy[END_REF][START_REF] Fragola | Apertureless scanning near-field optical microscopy in liquids : applications in fluorescence imaging[END_REF].

IR spectra of analytes and thin films have been recorded by combining a Fourier transform infra-red spectroscopy instrument with an AFM [START_REF] Hammiche | Photothermal FT-IR spectroscopy: A step towards FT-IR microscopy at a resolution better than the diffraction limit[END_REF][START_REF] Hammiche | Mid-infrared microspectroscopy of difficult samples using near-field photothermal microspectroscopy[END_REF]. This approach is based on an opto-thermal method that utilizes commercial AFM probes as temperature sensors which enabled measurements of opto-thermal signals induced by absorption of IR radiation [START_REF] Hammiche | Progress in near-field photothermal infra-red microspectroscopy[END_REF][START_REF] Reading | Micro-thermal analysis of polymers: Current capabilities and future prospects[END_REF]. This method enabled measurements of samples down to 5 ms in size. An alternative approach to opto-thermal based imaging, referred to as AFMIR, has enabled IR imaging of materials with sub-wavelength lateral resolution [START_REF] Dazzi | Subwavelength infrared spectromicroscopy using an AFM as a local absorption sensor[END_REF][START_REF] Dazzi | Analysis of nano-chemical mapping performed by an AFM-based ("AFMIR") acousto-optic technique[END_REF][START_REF] Houel | Ultraweak-Absorption Microscopy of a Single Semiconductor Quantum Dot in the Midinfrared Range[END_REF][START_REF] Mayet | Sub-100 nm IR spectromicroscopy of living cells[END_REF]. This method measures IR absorption directly via measuring local transient deformation in the AFM cantilever induced by an infrared pulsed laser tuned at a vibrational absorbing wavelength generating IR absorption spectral information.

AFMIR has been applied to image E. Coli bacteria at N-H resonance frequencies, expitaxial quantum dots in resonance with intra-sub band transitions and live cells monitoring the glycogen band centred at 1080 cm -1 with a sub-diffraction limited image resolution of 60-100 nm [START_REF] Dazzi | Analysis of nano-chemical mapping performed by an AFM-based ("AFMIR") acousto-optic technique[END_REF][START_REF] Houel | Ultraweak-Absorption Microscopy of a Single Semiconductor Quantum Dot in the Midinfrared Range[END_REF][START_REF] Mayet | Sub-100 nm IR spectromicroscopy of living cells[END_REF]. This experimental methodology requires the use of an attenuated total internal reflection (ATR) arrangement in combination with IR cyclotron radiation. This has effectively limited the application of the technique to samples of thicknesses on the order of the excitation wavelength due to the requirement of evanescent wave propagation through the sample. In order to study samples which are thicker alternative experimental designs are required. Here, we outline an experimental method for sub-micron IR surface imaging (referred to as s-AFMIR) that directly excites samples surface via a top-down excitation arrangement [START_REF] Hill | nano-Infrared surface imaging using an OPO and an AFM[END_REF]. Additional novel aspects of the outlined experimental approach include the use of an optical parameter oscillator (OPO) tuneable IR source, replacing cyclotron and black body radiation filaments as sources for IR radiation. This approach is different from other work on work combining AFM and optics to enable sub-diffraction IR imaging. Hillenbrand et al applied a method referred to as scattering-type scanning near-field optical microscopy (s-SNOM) to enable subdiffraction IR imaging [START_REF] Aizpurua | Substrate-enhanced infrared near-field spectroscopy[END_REF][START_REF] Cvitkovic | Material-specific infrared recognition of single sub-10nm particles by substrate-enhanced scattering-type near-field microscopy[END_REF][START_REF] Brehm | Infrared spectroscopic mapping of single nanoparticles and viruses at nanoscale resolution[END_REF]. In s-SNOM the metalized tip of an Atomic Force Microscope (AFM) is illuminated by laser light. The effect of this is to excite localized surface phonon-polaritons at the tip apex by the concentrated optical field. This produces a near-field resonance at a material specific frequency close to the LO phonon frequency. This method requires the AFM tip to oscillate and the scattered light is collected in the far-field. Aigouy et al also has applied a oscillating tip and far field light collection methodology to enable sub-diffraction optical imaging [START_REF] Formanek | Analysis of the measured signals in apertureless near-field optical microscopy[END_REF][START_REF] Fragola | Apertureless scanning near-field optical microscopy in liquids : applications in fluorescence imaging[END_REF].

The outlined method here contrasts with these approaches to perform sub-diffraction imaging by using the excitation light field to induce a topographic change in the sample, the intensity of which is measured as a function of wavelength in in order to construct an IR spectrum. This requires that the AFM tip be keep in contact with the sample and there is no need to collect the scattered light from the sample. This enables the measurement directly of light absorption. The scattering experiment provides less direct access to the IR absorption spectra of materials as the measured response is a convolution of this and the local dielectric properties.

Instrumentation

The s-AFMIR experimental set-up was based on an in-house built IR laser source and a commercial Atomic force microscope. Mid-IR radiation was generated using a periodically poled LiNbO 3 crystal emitting tuneable IR laser radiation, tuneable over 3.13 to 3.57 m as demonstrated by Vodopyanov [START_REF] Vodopyanov | Broadly tuneable noncritically phasematched ZnGeP2 optical parametric oscillator with a 2-mu J pump threshold[END_REF]. The OPO laser provides a high power infrared light source (compared to black-body sources). The OPO was constructed using an PPLN crystal (Super Optronics Inc) and a nanosecond Nd:YAG pump laser (model NL202, EKSPLA). The PPLN crystal possessing a period of 29.0-30.6 m fanning and was kept at a constant temperature of 90 0 C using a crystal oven heater (temperature controller, model TC038 and model III crystal heater, HC Photonics Corp). The OPO set up is outlined in Fig 1 . Selective filtering of laser radiation was performed using a long pass filter (L.P-2500 nm, Spectrogon).

Wavelength output was monitored using the frequency mixed component generated at the 800 nm region of the visible spectrum using an ocean optics fibre coupled CCD and spectrograph. The PPLN crystal was mounted onto a linear stage (Melles Griot) and was scanned with the wavelength output monitored. The output power was monitored to be c.a. 3 mW with pump power of c.a. 400 mW.

The IR laser is directed onto the surface of the sample been probed by the AFM tip using mirrors as outlined in Fig 1 . The excitation beam was focused using a convex mirror. Careful positioning of the excitation beam with respect to the tip was required to optimise the sample. The AFM (Veeco Explorer system) was used with a scanner with lateral and vertical scans of 100 x 100m and 10 m respectively. A Stanford SR650 programmable filter was used to amplify and filter the signal from the AFM.

The filtered signal was routed to the input of an Agilent DSO5012A oscilloscope where it was retrieved using in-house software and analyzed with the WSxM program [START_REF] Horcas | WSXM: A software for scanning probe microscopy and a tool for nanotechnology[END_REF]. Tips were silicon nitride v-shaped cantilever tips (used as received from Veeco).

The AFM is operated in contact mode, with a typical setpoint of the tip between 5 and 10 nN. Through analysis of the force curve of the contact and by establishing the setpoint, the AFM can remain in a linear range in contact with the surface in line with the literature [START_REF] Dazzi | Analysis of nano-chemical mapping performed by an AFM-based ("AFMIR") acousto-optic technique[END_REF].

Samples were prepared on CaF2 (Aldrich) or Mica substrates. Analytical grade glycerol (Aldrich), polystyrene (PS) films (Aldrich) and 3 m sized beads (Aldrich) were used. Polystyrene beads (PSB) were dispersed onto a mica surface and heated. This enabled the beads to be fixed to the substrate and are not free to be moved when interacting with the probing AFM tip. PS film was mounted directly onto the substrate. The PSB (on average 3 m in diameter) were purchased from Aldrich. A commercial Attenuated Internal reflection infrared (ATR-IR) spectrometer was used to record a reference IR spectrum of polystyrene.

Origin of s-AFMIR signal

The origin of the s-AFMIR comes from optically induced effects that are directly proportional to IR absorption. The AFM cantilever tip was positioned over the sample with the tip in contact with the sample surface (as outlined schematically in Fig 2).

Following the application of the IR radiation source the cantilever tip response was monitored. The specific response studied was a change in the vibrational motion of the tip arising from absorption of radiation by the sample. The energy absorbed is dissipated through thermal and acoustic mechanisms. Propagating acoustic waves create a deformation in the surface topography which can be detected by the AFM tip (see Fig 2). As an IR laser excitation source is tuned into resonance with a vibration mode, absorption of IR radiation increases and in turn the energy absorbed increases.

Recording the cantilever displacement intensity as a function of excitation wavelength enables either IR spectra or IR imaging to be performed when the AFM cantilever is stationary or is scanning respectively. The absorption of the excitation radiation exponentially falls as the excitation radiation propagates through the absorbing material [START_REF] Jung | Modelling and recording characteristics of a dye-doped polymer bilayer film[END_REF][START_REF] Subrahmanyam | Thermal expansion of irradiated polystyrene[END_REF]. As a consequence the applied energy from the excitation radiation will be converted into thermal and acoustic energy predominantly at and near the surface of the sample. The effective spatial resolution been a function of the sample under study. AFMIR has been applied to study quantum dots with a spatial resolution of 60 nm [START_REF] Houel | Ultraweak-Absorption Microscopy of a Single Semiconductor Quantum Dot in the Midinfrared Range[END_REF]. This indicates that the propagation lengths of the acoustic waves that are responsible for the absorption measurement mechanism are short enough in such materials to enable nanoscale imaging.

Studies of bulk material

Polystyrene films (20mm x 20 mm x 1 mm) were studied using s-AFMIR. The AFM cantilever tip was positioned over the centre of the PS film with the tip in contact with the sample. Following the application of the IR radiation source the cantilever tip response was monitored. Fig 3 shows an s-AFMIR spectrum recorded for a PS film, shown along with an IR spectrum recorded using a commercial IR instrument.

Inspection of the spectra show that both possess the same spectral features. This indicates that the s-AFMIR method has recovered the IR spectrum for a PS film with the characteristic vibration intensities arising from the aromatic ring, (C 6 H 5 ), giving rise to the group of bands above 3010 cm -1 and bands below 3010 cm -1 present arising from the saturated main chain CH groups.

Mesoscopic systems

Localised spectroscopy

Studies of individual PSBs were undertaken to demonstrate imaging small microscopic objects using this approach. A single PSB, 3.5 m in diameter and 750 nm in height was located using AFM topography scanning. The AFM cantilever tip was positioned over the centre of the PSB with the tip in contact with the sample.

Recording an s-AFMIR signal when the AFM tip is stationary enables an IR spectrum of a sample to be recorded as shown in while the ATR-IR based spectrum has a wavelength resolution of 1 cm -1 . The s-AFMIR resolution was due to the scanning increments. It is noted that the limiting resolution will be the PPLN IR source which is on the order of <6 cm -1 with the outlined experimental set up. In order to access this resolution the use of a precision controlled motorised stage or manual stage with micron resolution is required to scan across the PPLN crystal across the Nd:YAG pump laser beam. This must be coupled with precisely focused laser pump excitation beam that matches the resolution in the fanning gradient in the PPLN crystal, noting that the pump power must not exceed the damage threshold of the PPLN crystal. Analysis of the s-AFMIR spectrum of PSB in Fig 3 shows that there are differences in the spectral features when compared to the IR spectrum of the PS film. The band at 2960 cm -1 is larger in relative intensity in the PSB spectrum compared to the PS film. This spectral feature difference may arise from the local structural form that each of the polymers possess. Studies of PS with different morphologies has shown that the 2960 cm -1 band is sensitive to the presence of these morphology differences i.e. spherical beads or linear films of PS, it is noted that the heating of the PSB beads may also result in the formation of different polymer phases resulting in the observed relative intensity changes [START_REF] Duchet | Study of polystyrene microstructures and nanostructures synthesised in particle track-etched membranes used as templates[END_REF][START_REF] Vilaplana | Changes in the microstructure and morphology of high-impact polystyrene subjected to multiple processing and thermooxidative degradation[END_REF].

The AFMIR spectrum was recorded using the FFT of the cantilever oscillation as a function of excitation wavelength. The FFT of the signal shows the presence of numerous peaks corresponding to the different resonant vibrational modes of the AFM tip as a function of frequency. By monitoring the intensity of the FFT peaks it is possible to track the resonant response of the cantilever that is influenced by the absorption behaviour of the sample. A previous report by Hill et al outlined that s-AFMIR imaging showed that through the application of FFT signal processing the signal to noise ratio can be increased [START_REF] Hill | nano-Infrared surface imaging using an OPO and an AFM[END_REF]. The use of FFT signal processing creates a negligible background for the off-resonance signal making top-down s-AFMIR and ATR AFMIR methods comparable in regard to reduction in off-resonance background signal which demonstrates the high contrast of spectroscopic measurement capabilities in both approaches.

The image resolution of s-AFMIR was estimated to be 200 nm. Measuring a line scan across the edge of a PSB and comparing the s-AFMIR resolution with an AFM topography scan recorded at the same time estimated the s-AFMIR resolution to be 200 nm comparable with that of the AFM (see Fig 4). As show the greatest polystyrene mass to be present. Differences between AFM topography and s-AFMIR images which are present may arise from the presence of thermal drift of the mechanical device occurring during the duration of the measurement of s-AFMIR images i.e. 20 min. Analysis of the IR images shows that the left hand side of the PSB is more pronounced than the right hand side. This is potentially associated with the angle of incidence of the excitation laser. Previous AFMIR studies of E.coli [START_REF] Dazzi | Analysis of nano-chemical mapping performed by an AFM-based ("AFMIR") acousto-optic technique[END_REF] reported that the resultant IR images of the bacterium possessed weak intensity at centre of the bacterium and relatively intense signal at the edges. This was explained due to the bacterium scattering the incident electric field around the bacterium resulting in a lightening distribution and not a homogenous distribution. In additional it was noted that the signal is greater at one side than the other which was explained by referring to the angle of incidence of the excitation light. In order to overcome these inhomogeneous distributions requires careful choice of the angle of incidence of the excitation radiation beam. Never the less in the reported case, and in the images presented here, the IR images match very well with the AFM topography images. Analyses of smaller sample sizes were undertaken. Fig 5 shows an AFM topography image and an s-AFMIR image recorded on-resonance at 2965 cm -1 of moulded PSB formed by heating the sample. The AFM topography image shows a maximum height of 367 nm for the sample. The s-AFMIR image replicated the AFM topography features and demonstrates that thin surface features on the order of >367 nm can be imaged.

Sub-wavelength imaging

Potential improvements

Top-down excitation arrangement enables adequate excitation of a surface area of several microns using the out-lined set-up. The focusing of the excitation IR wavelengths via the use of an elliptical mirror practically enables focusing of the IR beam on the order of >10 microns. Focusing at the diffraction limit of /2 would enable a beam on the order of two microns to be achieved. This would limit s-AFMIR imaging to length scales on this order as imaging must occur within the IR excitation area. Imaging was performed without tightly focusing the excitation beam in order to excite a large area. This was done at the expense of signal sensitivity which would expect to improve with higher laser excitation power.

A number of improvements in the experimental system may lead to a further improvement in image resolution. Studies have shown that the tip motion is sensitive to both tip-surface and the tip-environment interactions [START_REF] Dazzi | Subwavelength infrared spectromicroscopy using an AFM as a local absorption sensor[END_REF]. This indicates that choice of tip composition should consider the material system been probed in order to optimise these interactions. This may include optimisation of AFM tip design to lead to more sensitive cantilever response in addition to reducing signal response arising from direct AFM tip excitation by the excitation laser. Studies of the effect of temperature on microcantilever resonance response showed that for mono-material cantilevers frequency varies in direct proportion to the temperature in line with the decrease in Young's modulus with increasing the temperature. When the cantilever is bi-material, the response is nonlinear due to differential thermal expansion. This indicates the choice of material used to build the tip with is important. Optimisation of AFM tip design may also include introduction of opto-thermal functionalism taking advantage of potential research advances in the development of opto-thermal IR imaging which will increase the range of measurements that can be made using the experimental approach outlined here.

Conclusion

A technique for sub-wavelength infrared surface imaging and spectroscopy of microstructured polymeric material has been demonstrated with an image resolution of /100. In this way the surface features of a sample can be chemical characterised via

Fig 3 .

 3 The spectrum is shown along with a reference IR spectrum of a polystyrene film, recorded using a commercial IR instrument for comparison. The s-AFMIR spectra shown in Fig 3 possess a wavelength resolution of 12 cm -1 ,

  Fig 4 and Fig 4's insert at a spatial separation of 200 nm the AFMIR signal be differentiated from each proceeding point producing a line-scan plot that was almost identical to that of the AFM.

Fig 5 shows

 5 Fig 5 shows IR images of a single PBS. Shown is an AFM topography image and IR

Fig 6

 6 Fig 6 shows the corresponding IR absorption images of the same bead recoded at five

Fig. 1 .Fig 2

 12 Fig. 1. Schematic drawing of a) periodically poled LiNbO3 crystal emitting tuneable

.

Fig. 4 .

 4 Fig. 4. a) Plot of PTIR (black curve) and AFM topographic(gray curve) data from a

Fig. 5 .

 5 Fig. 5. a) Images recorded from a single PSB, an AFM topography image (top), localised s-AFMIR absorption images recorded at 2920 cm -1 (on resonance) (middle)and an s-AFMIR image at 2965 cm -1 (off resonance) (bottom), shown also are

Fig. 6 .

 6 Fig. 6. s-AFMIR Images for a single polystyrene bead recorded at different excitation

inspection of the resulting IR spectra and images. This was achieved by combining a phase matched optical parametric oscillator laser as the IR source and an atomic force microscope as the detection mechanism. The technique uses a novel surface excitation illumination by focusing, at the diffraction limit, the excitation light directly onto the surface of the sample in presence of the AFM tip. This method enables chemical mapping and AFM topography imaging to be performed simultaneously with an image resolution of 200 nm.