A LabVIEW based microscope for Multi-photon excited fabrication

Paul J. CampagnolaUniversity of Connecticut Health Center

Department of Cell BiologyCenter for Biomedical Imaging Technology

AcknowledgmentsDr. Swarna Basu

Ms. Mallika Sridhar

NIH (NIBIB)

Outline

1) Introduction to multi-photon excitation/fabrication

2) Design, Construction and characterization of new LabVIEW based microscope

3) Diffusion within MPE Cross-linked Protein Matrices:normal and anomalous

4) Enzymatic Activity within MPE Cross-linked Matrices:maintain biological activity

iomedical Applications of Optical Fabrication

Nano-structured and/or Biomimetic Biomaterials

Tissue Engineering Scaffolds

Drug Delivery Systems

Minimally Invasive Tissue Repair

Adding Bio-functional Groups to Chips

ome Available Fabrication Methods

hotolithography

200 nm features sizes

essentially 2-dimensional

not bio-compatible

icrocontact printing (stamping)

1 micron feature sizes

essentially 2-dimensional

limited chemistries (thiols)

One and two photon absorption physics

Requires high power:Confines excitation toPlane of focus

Two-photon images layer by layerCan also fabricate layer by layer

Multi-Photon Excitation [Rose Bengal]

S0

S1

S2

T1

hν=800 nm

10-12 s

Mixture: “one pot”•polymer, protein•Photoactivator: Rose Bengal•buffer

Crosslinking proceedsVia singlet oxygen

First layer on glassBuild successive layers

dvantages of Multi-photon Excitation in Fabrication

Intrinsic 3-Dimensionality, Freeform

Excellent Lateral (X,Y) and Axial (Z) Resolution

Large Depth for Fabrication (reduced Rayleigh Scattering)

Little Near Infrared and IR Absorption of

Biomolecules Minimizes Out-of-Plane Photo-Damage

IR Light Eliminates Problems with UV Excitation

UV Optics

UV Lasers

Fluorescein excited by two-photons at 700 nm

Fabrication of Complex 3-D structures (SEM)

TMPTA

BSA

Bovine Serum Albumin

Trimethyolpropane Triacrylate

Alkaline Phosphatase in BSAELF fluorescence activity assay

~350 nm

10 um

Minimum Observed Feature Sizes (nm) for TMPTA (800 nm)

NA lens 2-photon* 3-photon* Predicted*0.5 2944 966 976

0.75 1191 606 650

1.3 735 344 375

NAd

222.1

minλ

=

*Prediction based on 1-photon 800 nm Abbe′ Limit

Missing “resolution?”

Synthetics can propagate outside focal zone: chain reaction

P t i li it d t f l

NA=numerical Aperture

New Multi-Photon Excited Fabrication Instrument/Microscope:

• Laser, Upright microscope

•Closed Loop Galvos scanning mirrors

•One DAQ Board (6042E) entirely interfaced with Labview

• Controls Galvo scanning

• Synchronous Fluorescence Diagnostics/Imaging (existing designs used two boards)

GUI allows choice of• scan size, pixel density• shape : rectangle, circle, ellipse (filled in, perimeter)• x,y aspect ratio• speed

Scan, image 500 x 500 frame /secondComparable to commercial instruments

Control Software Logic Scheme

Initialize Analog Outputof the DAQ board

Initialize the Counter

Display Image

Shutdown

Save Image

Set up X and Y vectorsfor Analog Output

User Input:X pixels,Y pixels,

Type of Scan,X and Y Amplitude

Calculate RampFunction for 1

line RasterSetup X Axis Vector

for 1 Page

Set up Y Axis vectorto step down after

each raster line of X

Configure theanalog output

channels through‘AOCONFIG’

Write thepre-calculated vector

into the bufferthrough ‘AOWRITE’

Setup a connectionbetween Analog Output

Update Clock anda PFI line

Configure Counter 0for ‘Buffered Period

Measurement’ through‘GROUP CONFIG’

Setup the size ofthe Counter Bufferthrough ‘BUFFER

CONFIG’

Define the source ofthe Counter

(Discriminated Outputfrom a Photo multiplier)

Define the Gateof the Counter

Start the Analog Outputoperation through

‘AOSTART’withuser specified Update Rate

Collect the counter’s data (Itcounts the Number of TTLpulses received from the

source between 2 gating pulses)

Convert the Counter Outputto an Intensity Graph to

represent the florescenceof the sample area scanned

Clear Analog OutputBuffers

Set both channelsto 0 volts

ResetCounter

Save image to aJPEG or

BMP format

The Gate of the counterand the Update clock are connected

to achieve synchronization

Synchronize, Scan andAcquire

Analog Output feedsthe servo motors which drives

the galvanometers

Counters: synchronizationand data acquisition:

Single Photon countingD/As provide x,y ramps:To servo controllers

Multi-photon Excited Fabrication Instrument

x y

LWP DM

ScanningGalvos

Sample

HR

Filters

PolarizerTransmitted Light orTPEF orSHG Detection

vis nir

0.9 NA

TPE FluorescenceDetection

modelocked ti:sapphire

5 Watt Nd:YVO4

PMT2

PMT1

650 nmlong pass filter

700- 9400 nm100 femtoseconds76 MHZ

RecollimationLenses

525 nm Photon Counting

Photon

Counting

DAQ

900 MHz PC

ServoControl

ND Filters

L1

L2 L3

Pupil Transfer Lens

UprightMicroscope

Broadband ornear UV

More flexible scanningThan confocals

Synched fluorescence, SHGFor imaging, diagnostics

RSI, 2003

Data collection usesSingle photon counting

0.1 0.2 0.3 0.4 0.5 0.60.0

500

400

300

200

100

0

Analog Voltage

Fiel

dSi

ze(m

icro

ns)

R2 =0.991

y=694.74x + 24.4

30 microns

Field Size Calibration for 20x Lens

15 micron beads

(Dye labeled)

Need plot for every objective

Optical Resolution of New Microscope

Imaging sub-resolution 100 nm fluorescent beadsto obtain Point Spread Functions (PSFs) at 1.3 Numerical AperturePSF= image from point source of lightFit data to Gaussian Profile

Both lateral and axial agree well with theory

Minimum Fabricated Feature Sizes in new microscope at 0.75 NA

Lateral “Resolution” Axial “Resolution”Height ~ 2microns

Determined by imaging edge at 1.3 NA

Scales right with High resolution PSF data

Also good agreementwith theory

Scanning Transmitted Light Image of Stepped Pyramid of TMPTA

Progressively smaller layers built on top of each other

Two-photon Excited Fluorescence Image of BSA Channel Structure

Potential in Microfluidics Applications

Flow on bio-”chips”

TPEF Images of Box Structures from TPMTA

User-defined size, aspect ratio

Could created by tedious shuttering and page scanningDirect scanning much more efficient

Applications for cell encapsulationAdd reagents, drugs, growth factors

Control of Porosity

TMPTA BSA AP/ BSA

10 µmTMPTA = Trimetholpropane Triacrylate

BSA = Bovine Serum Albumin

AP = Alkaline phosphatase

Sustained Release of Model Pharmaceutics:Rhodamine 610 from Acrylamide Matrices

Time (minutes)0 20 40 60 80 100

Nor

mal

ized

Flu

ores

cenc

e In

tens

ity

NR=50 10 minutes

NR=7531 minutes

Measure diffusion by disappearanceOf fluorescence from R610

Tighter crosslinking: slower release

Increase crosslink densityby increasing number of scans

Release is found to be diffusion limited, i.e. ~ linear with the square root of the release time.

Diffusion within MPE Crosslinked Matrices?

Tracer Fluorescent Dyes

Dextrans are high molecular weight (10 and 70 kD) conjugatesSugar groups are hydrophilic

Measure Diffusion by FRAP in BSA Matrices

Texas Red (Sulforhodamine 101) in BSA Matrix

Point bleach, line scan

D

Dτ

ω8

2

=Diffusion Coefficient ω=beam waist

~100-1000 fold slower than solution

Tuning diffusion coefficient through 10 layer BSA structure

1 micron layers3 micron Axial PSF

Little overlap attop, bottom

Can achieve fairly uniform diffusion by optimizing power

Diffusion of Rhodamine dyes within Crosslinked BSA matrices

Diffusion approximately scales as m1/3

Like simple spheresAsymptotic diffusionAt high crosslinking:Used all reactive sites

Normal vs Anomalous Diffusion

Important in Cell Biology

•Collisions with cytoplasmic proteins

•Obstacles:intracellular organelles, cytoskeletal elements

•Potential traps: Hydrophobic, hydrophilic interactions

Simple Manifestation: Mass scaling greatly deviates

Hard to study directly in cell biology,Fabricated protein matrices good model system:Change chemistries of tracer dyes

Diffusion of Dye- Dextran Conjugates in BSA

⎟⎟⎠

⎞⎜⎜⎝

⎛−−−=

D

tCCCCτ

exp)( 0maxmax

( )max max 0d

tC C C C expα⎛ ⎞⎛ ⎞

⎜ ⎟= − − −⎜ ⎟⎜ ⎟τ⎝ ⎠⎝ ⎠

Normal Diffusion

Anomalous Diffusion

α<1 is anomalous

Diffusion is normal:α∼1 for both dyesTexas Red more hydrophobic than RhodamineBut high molecular weight hydrophilic dextran dominates

Diffusion of different Texas Red Dyes

α=0.95α=0.6

α=0.5

Diffusion of 10 kD Dextran is much faster than Unlinked Texas Reds! highly anomalousDue to Hydrophobic chromophore and hydrophobic Protein

Normal and Anomalous Diffusion and Dye Concentration

Concentration Independent: Normal diffusion: RhodamineStrong Dependence:Anomalous Diffusion of Texas Red

Alkaline Phosphatase (metalloenzyme)Dimer of 70 kD monomers

Active site

Active site

Active Enzyme Bound in Protein Gels: Alkaline Phosphatasein an AP or BSA Matrix

N

NH

OCl

Cl

OPO

OON

NH

OCl

Cl

OH

Alkaline Phosphatase

“ELF” substrateSoluble in waterWeak blue fluorescence

Insoluble in waterIntense green fluorescence

Rise in Fluorescence After ELF Addition

Time (Minutes)0 2 4 6 8 10 12 14 16

Fluo

resc

ence

Inte

nsity

(Arb

itrar

y U

nits

)→

Relative Enzyme Reaction Rates and Crosslink Density:AP and BSA matrices

Higher crosslinking: slower diffusion

Similar Reactivity:No apparent denaturing at shorter wavelength

MPE at 750 nm shown to be damaging in live cell imaging:Highly nonlinear effects

Michaelis Menten Enzyme Kinetics

MKSSVV

+=

][][

max KM= substrate concentration at Vmax/2ELF substrate

][max

EV

kcat = kcat is turnover rate

•Measure V at several [S] i.e. ELF concentrations

•Plot 1/V vs 1/S to obtain KM and then kcat

•kcat/ KM is specificity constant

•Compare to known literature values for activityTo determine if enzyme is denatured

Data Acquisition for Michaelis Menten AnalysisCrosslinked Alkaline Phosphatase

MM Form 1/v vs 1/s

kcat/KM= 1.3 x105 M-1s-1

Good agreement withKnown valuesEnzyme is active

Alkaline Phosphatase KineticsIn different matrices

BSA/AP 25% Acrylamide

kcat/KM= 1.0 x105 M-1s-1 kcat/KM= 2.0 x106 M-1s-1

Faster diffusion in the acrylamide hydrogel

Bound vs Entrapped Alkaline Phosphatasein protein and polymer matrices

1) AP in AP and AP/BSA protein matriceshigh salt extractions and ELF analysis (inside and out)lower bound of 90% covalently bound in matrix

2) AP in polyacrylamide matrixhigh salt extractions and ELF analysisnone left in matrix recovered AP still active

Two different matrix environments afford different molecular characteristics for diffusion and reactivity

Conclusions and Future Directions

1) Constructed Low-Cost Microscope/Fabrication Instrument

2) Optical Performance is matches theoretical predictions

3) More versatile than commercial instruments

4) Next Generation: Integrate CAD for true freeform capabilities