Located in the Science Lab Bldg, College & Fell Av., Normal IL 61790

Contact Kevin Edwards for confocal questions or assistance.

ISU is pleased to announce the opening of our new Confocal Microscopy Core Facility. It is funded by a grant from the National Science Foundation. See the award page for more details

Components and capabilities of the ISU Confocal Facility

Leica TCS SP8X-FALCON Confocal Microscope (2019-). This core facility is available for users internal or external to ISU.

Microscope:

  • DMi8 CS inverted microscope; DIC
  • Air Table / Compressor
  • Super Z Galvo Stage; optional Universal or Multiwell plate inserts
  • LMT200-T high precision scanning stage
  • Adaptive Focus Control
  • Turret Cooling
  • LED brightfield light source
  • Lumencor SOLA SE II white LED light source for wide-field fluorescence
  • Leica DFC7000 T cooled color camera for non-confocal light and fluorescence imaging
  • Transmitted light PMT
  • Blue/green/red/far red filter cubes for wide-field fluorescence:
    • DAPI bandpass filter cube. Excitation: BP 350/50, Emission: BP 460/50
    • LED 470 filter cube. Excitation: 470/40, Dichroic: 495, Emission: 525/50
    • RHOD bandpass filter cube. Excitation: BP 546/10, Emission: BP 585/40
    • Y5 bandpass filter cube. Excitation: BP 620/60, Emission: BP 700/75

OBJECTIVES:

Summary: 5x; 10x; 20x-imm; 40x-oil; 40x-water; 63x-oil; 63x-glyc; 100x-oil.

  • 1) HC PL FLUOTAR 5x/0.15. For use with and without coverglass, no immersion. Free working distance: 12.0 mm.
  • 2) HC PL APO 10x/0.40 CS2. For use with 0.17 mm coverglass. Air immersion. Free working distance: 2.2 mm.
  • 3) HC PL APO 20x/0.75 IMM CORR CS2. Correction collar for use with and without coverglass and use with water, glycerine and oil immersion. Free working distance: 0.67 mm (water immersion and 0.17 mm coverglass).
  • 4) HC PL APO 40x/1.30 Oil CS2. Oil immersion. Free working distance: 0.24 mm.
  • 5) HC PL APO 40x/1.10 W motCORR CS2. Motorized correction collar for adjustment of coverglass thickness from 0.14-0.18 mm, water and saltwater immersion and within the temperature range 20-40°C. Free working distance: 0.65 mm.
  • 6) HC PL APO 63x/1.40 OIL CS2. Oil immersion. Free working distance: 0.14 mm.
  • 7) HC PL APO 63x/1.30 GLYC CORR CS2. Glycerol immersion. Correction collar for use with coverglass (0.14-0.19 mm), glycerol immersion (80% glycerol / 20% water) and within the temperature range 20-40°C. Free working distance: 0.30 mm.
  • 8) HC PL APO 100x/1.40 OIL CS2. Oil immersion. Free working distance: 0.13 mm.

TCS SP8 Confocal Scan Head

  • Tandem Scanner
    • Non-resonant (conventional/variable) Scanner: 1 - 1800 Hz (7 fps @ 512 x 512 px, 84 fps @ 512 x 16 px), line frequency up to 3600 lines/second (bidirectional), max. scan format 8192 x 8192 px, scan field 22 mm diag.
    • Resonant Scanner (ultra-fast, reduced photodamage): 8000 Hz (28 fps @ 512 x 512 px, 290 fps @ 512 x 16 px), line frequency up to 16,000 lines/second (bidirectional), max. scan format 2496 x 2496 px, scan field 13 mm diag. Optical field rotation 200°.
  • Notch filter set (provides additional suppression of excitation light) - NF 445; NF 488; NF 514; NF 458/514; NF 488/561/633; NF 594; NF 445/594

  • 5-channel filter-free prism detector system. Emission bands freely tunable with edge-positioning precision of 1nm. Lambda-Scan mode permits recording of spectral image series.
    • Two PMTs (Hamamatsu R 9624 photomultiplier/ 40 MHz sampling/ detection range 400 - 800 nm)
    • Three HyD hybrid Spectral detectors. Very low dark noise; Photon counting capability; Enables time gated detection.
  • Programmable control panel

Excitation

  • Continuous wave lasers
    • 405 nm Diode Laser (50 mW), DMOD
    • Argon Laser (65 mW): 458 nm, 476 nm, 488 nm, 496 nm, 514 nm
  • Pulsed lasers
    • White light laser WLL2. Select up to 8 lines, individually tunable in steps of 1 nm from 470 to 670 nm, ~1.5 mW power per line. Pulsed, 80 MHz. 8 channel AOTF (Acousto-Optical-Tunable-Filter) for rapid modulation of laser intensity.
    • Recording of two dimensional excitation-emission spectra supported by software for setup and display of 2D spectra.
    • Lightgate function for avoidance of reflected laser light
    • Pulsed 440 nm laser, LDH-P-C-440B. Suited to lifetime based measurements including FLIM. Software controlled selection of pulse frequency (40, 20, 10, and 5 MHz).
  • LAS X Lightning Expert software for adaptive deconvolution

  • FALCON (FAst Lifetime CONtrast) real time FLIM detection; FLIM- FRET analyzer.

  • HPZ840 Workstation with Windows 10 Professional (64 bit) operating system. 2x Intel 4-core Xeon E5-2637 V4 3.5 GHz. 128 GByte RAM. NVIDIA Quadro GP100 16GB Reference GPU (3584 cores).
  • Leica LAS X system software; LAS X MicroLab
  • Software modules: LAS X 2D Analysis/Multi Channel; LAS X 3D Visualization; LAS X 3D Analysis

Leica TCS SP2 Confocal Microscope (2004-). This is maintained for overflow use and for teaching/training purposes.

Microscope: Inverted Leica DM IRE2 HC Fluo TCS 1-B

  • Outfitted for DIC optics and standard fluorescence
  • Galvanometer driven z-stage
  • Trinocular head and color CCD camera, allowing for capture of nonconfocal images

Objectives

  • HC PL APO 20x/0.70 Multi-immersion- High numerical aperture Plan Apochromat Immersion lens for Water, Glycerol or Oil Immersion.
  • HCX PL APO 40x/1.25-0.75 Oil- High numerical aperture Plan Apochromat lens for Oil Immersion.
  • HCX PL APO 63x/1.4 - 0.6 Oil- High numerical aperture Plan Apochromat lens for Oil Immersion.
  • HCX PL APO 63x/1.20 W Corr/-CS- High numerical aperture Plan Apochromat Immersion lens for Water Immersion with correction collar. For use without coverglass.

Confocal Scan head: TCS SP 2 Basic Module with spectral detection.

Features:

  • 3 Laser sources:
    • Red (HeNe 633 nm)
    • Light Green (561 nm DPSS)
    • Blue (Ar 458-476-488-514 nm)
  • 6 channel Acousto Optical Tunable Filter (AOTF) for laser selection and attenuation.
  • Three channels for confocal fluorescence or reflected light.
  • Low-noise photomultipliers, 8 or 12 bit digitization
  • Transmitted light detector for recording bright field images
  • Spectral range of detector optics 400-800nm
  • Adjustable pupil illumination using Beam Expander
  • Variable, computer controlled detection pinhole diameter
  • Scan resolution up to 4096 x 4096 pixels
  • Zoom 1-32x
  • Scan rotation -2+92 degrees
  • Scan field diagonal 22mm (intermediate image plane)
  • LCS Leica Confocal Software incl. 3D, Physiology, & Multicolor packages.

Schedule: Once you are approved to use the confocal, Dr. Edwards will provide a link to the online reservation calendar (contract him via e-mail).

Resources: The following faculty can be consulted for advice on fluorescence techniques and imaging:

  • Edwards: general confocal information; Drosophila; arthropods; fluorescent protein methods
  • Gatto: cell culture; biochemistry
  • Hammond: fungal samples; fluorescent protein methods
  • Kirik: plants; live imaging
  • A. Mortimer: Drosophila
  • Sedbrook: plants; live imaging
  • Stein: neural imaging
  • Vidal-Gadea: C. elegans; neural imaging
  • Vogel: FACS; mouse tissues; immunological methods

Schematic diagram of the SP2 scan head: confocal