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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