Resources

Click here to download the Work Order

Confocal Imaging Core Contacts:

Director:
Sarah L. Dallas, Ph.D., Lee M. and William Lefkowitz Endowed Professor,
UMKC Department of Oral and Craniofacial Sciences
(816) 235-6295
dallass@umkc.edu

Co-Director
LeAnn Tiede-Lewis, Ph.D. Research Assistant Professor
UMKC Department of Oral and Craniofacial Sciences
(816) 235-2089
tiedelewisl@umkc.edu

Confocal and Multiphoton Microscopy

Our goal is to support the research programs of a diverse group of NIH funded and other users located in the UMKC Schools of Dentistry, Pharmacy, Biological Sciences and Nursing as well members of the UMKC Center of Excellence in the Study of Dental and Musculoskeletal Tissues (CEMT), and other researchers at UMKC. This confocal microscopy core resource is also available to the wider academic and commercial community in the Kansas City area. We are capable of accommodating a wide range of high-resolution microscopy project applications and fluorophores.

Features at a Glance:

  • Leica TCS SP5 II confocal on an inverted microscope platform
  • Leica TCS SP8 multiphoton on an upright microscope platform
  • Life Imaging Systems Incubation Chamber with 5% CO2 capability
  • Conventional confocal  and multiphoton microscopy of:
    • Fixed cells and tissue
    • Live cells and specimens
    • Live animals
  • Multispectral imaging
  • Fluorescence Recovery after Photobleaching (FRAP)
  • Optical Sectioning /3D Imaging
  • Second Harmonic Generation Imaging
  • High speed imaging for live cell imaging and imaging of delicate fluorophores
  • Imaging in Z scan mode (real time vertical sections)

Links and Downloads

Confocal Microscope Information

Microscope Information

 

Specifications at a glance

  • 2.5x, 5x, 10x, 20x, 25x 40x, and 100x objectives
  • Excitation Laser wavelengths ranging from 405 nm-633nm.
  • Conventional and Resonant Scanners for scan speeds ranging from 400 Hz-8000 Hz.
  • Leica SP detector employing prism and slider separation of emission wavelengths from 400nm-800nm with customizable bandwidths.
  • 3 Standard PMTs and 2 HyD PMTs
  • Galvo-Z Stage for fine controlled z positioning and Z scan imaging
  • Applications include: Live Cell Imaging, Intensity based imaging of a variety of fluorophores, co-localization studies, spectral imaging, time-lapse imaging, 3-D imaging, x-y tiling, mark and find, FRAP

Objectives

Objective Immersion
Type
NA Pixel Resolution (green) Working Distance
2.5x Fluorite Plan Air .07 2 µm 11.2 mm
5x  Plan Fluorite Air 0.15 920 nm 14.0 mmm
10x Plan Apochromat Air 0.4 350 nm 2.2 mm
20x Plan Apochromat Air 0.7 200 nm 0.59 mm
25x Plan Apochromat Water .95 150 nm 2.5 mm
40x Plan Apochromat Oil 1.25 110 nm 0.24 mm
100x Plan Apochromat Oil 1.44 95 nm 0.10 mm

Lasers

Laser Wavelengths
(nm)
Maximum Power
(mW)
Max Percentage
Advised
UV diode 405 50 15%
Argon Ion 458, 488, 476, 496, 514 Variable 15-30%
HeNe (green) 543 1 50-60%
HeNe (yellow) 594 2 50-60%
HeNe (red) 633 20 15%

Scanners

The availability of both a conventional confocal scanner and a resonance scanner allows the user to optimize for resolution or speed.

Scanner Frame
Scan
Speed
Line
Scan
Speed
Frame Rate
(512×512)
FOV Max
Image
Size
Beam
Park
Spot
Collection
Rate
Conventional 10-1400 Hz 100-2000 Hz 2/sec 22 mm- 3 mm 9600x 9600 yes 40 MHz
Resonance 8000 Hz 16,000 Hz 25/sec-200/sec 15 mm 1024x 1024 no N/A

Detection

AOBS: The use of an Acousto-Optical Beam Splitter in place of a traditional beam splitter allows for programmable, high-transmission direction of collected light without purchase of wavelength specific dichroics. The use of AOBS technology also enables non-distortion emission spectra to be used in many applications.

Prism and Sliders

The Leica SP5 employs a prism and tunable sliders to separate the collected light into channels for detection at the PMTs. Allows users to “dial in” their spectral collection window without the need for specific dichroic filters for each dye.

PMTs

Detector Number
Available
Quantum
Efficiency
(500nm)
Contrast
(A.U.)
(500 nm)
Bright R Bit Depth Photon
Counting
Mode
Standard 3 25% 5 No 12-16 No
HyD 2 45% 160 Yes 12 Yes

Automated Stage

Our system features a fully automated stage for movement in all three dimensions. X-Y tiling and mark and find features in the software can be automated to minimize the time spent with the microscope during image acquisition and to allow repeated imaging of many regions at timed intervals.

The Galvo-Z stage allows for fine z-plane adjustment necessary to perform 3-D reconstructions of samples within the focal resolution of the microscope. Adaptive focus features in the software allow for consistent maintenance of the z-position of the stage over long time scales.

Imaging Applications

Live Cell Imaging: Our confocal microscope is contained within a temperature controlled module with an attachment that allows for 5% carbon dioxide maintenance during imaging.

Intensity based imaging is available for all fluorophores and spectral imaging with appropriate excitation wavelengths.  Emission filters and bandwidths are not a concern with the Prism and Slider system of the Leica SP5.

Co-localization studies: Limited only by the resolution of the microscope set up chosen and the separation of the emission spectra of the fluorophores.  Up to five colors in a single scan are possible with options to use multiple (separate) scans to prevent bleedthrough.

Time-lapse imaging: system automation allows for time lapse imaging in a variety of applications for short term imaging and extended imaging periods

X-Y tiling, mark and find: Can be applied in conjunction with the majority of applications.

3-D imaging: use of the Galvo-Z stage and processing software allows for reconstruction of 3-dimensional images of samples.

FRAP: By parking the beam using the conventional scanner features of the microscope, spatially targeted photobleaching can be performed.

Multiphoton Microscope Information

Specifications at a glance

  • 10x, 25x 40x, and 63x objectives (others may be available on request from the confocal microscope)
  • Excitation Laser wavelengths 690-1040nm (multiphoton) 488nm, 552nm (for confocal mode).
  • Conventional and Resonant Scanners for scan speeds ranging from 400 Hz-8000 Hz.
  • Non-descanned dectection (multiphoton) using filter cubes DAPI/FITC, FITC/TRITC or CFP/YFP
  • Descanned (confocal) detection uses a prism and slider separation of emission wavelengths from 400nm-800nm with customizable bandwidths.
  • Two non-descanned HyD PMTs,  1 standard PMT for confocal detection
  • Applications include: Invtravital Imaging, Live Cell Imaging, Second Harmonic Generation imaging, Intensity based imaging of a variety of fluorophores, spectral imaging, time-lapse imaging, 3-D imaging, FRAP

Objectives

Objective Immersion
Type
NA Pixel Resolution (green) Working Distance
10x HC IR Apochromat Dipping Water 0.3 480 nm 3.6mm
25x Fluortar Dipping Water 0.95 150 nm 2.5mm
25x Plan IR Apochromat Water .95 150 nm 2.5 mm
40x Plan IR Apochromat water 1.10 130 nm 0.65 mm
63x Plan Apochromat Dipping water 0.90 160 nm 2.2 mm

 

Laser Wavelengths Maximum Power
488 nm solid state 488 20 mW
552 nm solid state 552 20 mW
MaiTai HP DeepSee 690-1040 1.5 W (average at 800nm)

Lasers

Scanners

The availability of both a conventional confocal scanner and a resonant scanner allows the user to optimize for resolution or speed.

Scanner Frame
Scan
Speed
Line
Scan
Speed
Frame Rate
(512×512)
FOV Max
Image
Size
Beam
Park
Spot
Collection
Rate
Conventional 10-1400 Hz 100-2000 Hz 2/sec 22 mm- 3 mm 9600x 9600 yes 40 MHz
Resonant 8000 Hz 16,000 Hz 25/sec-200/sec 15 mm 1024x 1024 no N/A

Non-Descanned Detection

For high sensitivity multiphoton signal acquisition, non-descanned detectors are used in conjunction with filter cubes for maximal light gathering with minimal light dispersion.  Current filter cube configurations are listed in the table.

Cube Dichroic (Beam Splitter) Filter 1 Filter 2
DAPI/FITC RSP 495 460/50 525/50
FITC/TRITC RSP 560 525/50 585/40
CFP/YFP RSP505 483/32 535/30


Descanned Detection

AOBS: The use of an Acousto-Optical Beam Splitter in place of a traditional beam splitter allows for programmable, high-transmission direction of collected light without purchase of wavelength specific dichroics. The use of AOBS technology also enables non-distortion emission spectra to be used in many applications.

PMTs

Detector Number
Available
Quantum
Efficiency
(500nm)
Contrast
(A.U.)
(500 nm)
Bright R Bit Depth Photon
Counting
Mode
Standard (descanned) 1 25% 5 No 12-16 No
HyD (Non-descanned) 2 45% 160 Yes 12 Yes

Automated Stage

Our system features a fully automated stage for movement in all three dimensions. X-Y tiling and mark and find features in the software can be automated to minimize the time spent with the microscope during image acquisition and to allow repeated imaging of many regions at timed intervals.

Imaging Applications

Intravital Imaging: Superior signal penetration of the long wavelengths used in multiphoton microscopy make the SP8 multiphoton ideal for intravital imaging with the proper specimen holder/ stereotaxic device and an IACUC approved protocol.

Live Cell Imaging: Our multiphoton microscope is contained within a temperature controlled module that has an attachment that allows for 5% carbon dioxide maintenance during imaging.

Intensity based imaging is available for all imageable fluorophores and spectral imaging with appropriate excitation wavelengths.

Time-lapse imaging: system automation allows for time lapse imaging in a variety of applications for extended periods

X-Y tiling, mark and find: Can be applied in conjunction with the majority of applications.

3-D imaging: Processing software allows for reconstruction of 3-dimensional images of samples.

FRAP: By parking the beam using the conventional scanner features of the microscope, spatially targeted photobleaching

Second Harmonic Generation Imaging: Imaging of highly ordered structures such as collagen or elastin is possible without the addition of dye or fluorescent tagging using natural birefringence.

Information for New Users

Registration and Training of Users:

  • Contact Dr. Tiede-Lewis or Dr. Dallas for a consultation to determine whether technician assisted operation, independent operation or advanced user operation is best suited to your experimental needs.
  • Fill out initial work request form (signed by PI) to register as a user.
  • Schedule and complete training.
  • Demonstrate competency for Independent User status.
  • For Advanced users log required hours and demonstrate higher level of competency.

Usage types 

Fee rates below are for UMKC personnel – for complete fee structure see below in Fees section

Designation Required
Training
Confocal Rate Multiphoton Rate Hours ofUse
Assisted Basic $40.00 $55.00 8a.m.-5p.m.
Independent Basic  competency $25.00 $35.00 8a.m.-5p.m
Advanced Independent +20 hrs $15.00-$25.00 $25.00-35.00 Open**


Scheduling of Use

  • Submit request for reservation to Dr. Tiede-Lewis.
  • Request will either be approved or denied based on order received and allocated usage.  Special consideration will be made to try to accommodate grant applications and pending manuscripts.

Biosafety, IRB and IACUC Approval

As part of the user registration process, biosafety concerns will be reviewed with the PI and appropriate IRB, biosafety or IACUC approval will be confirmed. Before any biohazardous samples are viewed on the confocal microscope, appropriate biohazard containment protocols must be in place and approved by the UMKC Biosafety committee.

Radiation

The confocal microscopy core is not approved to handle radioactive materials and cannot accept any such samples

Fees

Hourly Fees for Usage of the Leica TCS SP5 II Confocal Microscope

User Category UMKC Academic (non-UMKC) Commercial
Assisted $40.00 $50.00 $120.00
Independent $25.00 $35.00 $80.00
Time series*/Off peak** $15.00 $20.00 n/a

*Time series extending more than 4 hours
** Off Peak hours will be defined as ending at 10 am and beginning after 4 pm.

Hourly Fees for Usage of the Leica TCS SP8 Multiphoton Microscope

User Category UMKC Academic (non-UMKC) Commercial
Assisted $55.00 $65.00 $180.00
Independent $35.00 $45.00 $110.00
Time series*/Off peak** $25.00 $35.00 n/a

*Time series extending more than 4 hours
** Off Peak hours will be defined as ending at 10 am and beginning after 4 pm.

New or young investigators who do not yet have funding and are applying for NIH funding may apply for a fee waiver for an initial period of use of the microscope not to exceed 20h in order to generate preliminary data for grant applications.

Useful Information

Useful Information

 

Tips

For best results in fixed slides use a Toluene free nail polish such as “Wild Shine” to seal cover slips on slides.

Check fixation method for your fluorophores and samples. Some fluorophores and tissues require special fixation considerations.

Table of Common Fluorophores:
http://dbc.bio.uci.edu/OBCresources/fluorophores.pdf

Table of Fluorescent proteins:
http://www.microscopyu.com/articles/livecellimaging/fpintro.html