Microscopy & Histology

Microscopy & Histology

Overview

The Microscopy and Histology Core Facilities at La Jolla Institute were developed to help local scientists with their imaging needs in a static or dynamic (in vitro/in vivo) scenario by offering histology and microscopy services and providing support and training to interested researchers. Dedicated personnel can assist in all essential steps of an imaging experiment starting from experimental design, sample preparation through image acquisition and data analysis. The facility offers a range of light microscopes, related imaging systems and image analysis resources for assisted or independent use (upon training). The facility operates on a fee for use basis and is open to all potential users.

Services

From whole animal imaging to resolving details beyond the diffraction limit, LJI microscopy facility offers one stop shop for all your light microscopy needs. In addition to microscopy services, our staff offers full service histology. Dedicated personnel will take care of your samples to deliver high quality sections which can be digitized and analyzed with the help of our in-house experts. We offer advice on proper sample fixation, processing, embedding of tissues with attention to desired orientation, decalcification of bone tissue, cutting of paraffin blocks, cryosectioning, routine, special and immunostains as well as training in histological techniques.

Instrumentation

LEICA SP8 INTRAVITAL MULTIPHOTON/CONFOCAL SYSTEM

Capabilities

- Deep tissue imaging of live and fixed specimens with low phototoxicity and photobleaching

- In vivo (mouse) and in vitro (cultured cells, explanted organs) microscopy

- Enhanced resolution confocal and multiphoton microscopy

- 3D volume scanning, time lapse and automated multi-region imaging

Location

BSL2 area with badge-restricted access

System specifications

Microscope (Upright DM6000 CFS stand)

  • DM6000 CFS with motorized focus drive for transmitted light, epifluorescence and confocal laser scanning microscopy
  • Motorized Moveable Base Plate - Scientifica stage for ample space for the sample
  • 2 position motorized nosepiece
  • 6 position motorized nosepiece
  • Objective slider for 20x objective with motorized correction collar
  • Nosepiece piezoelectric actuator for a precise control of Z positioning

Scanner

  • Tandem (conventional and resonant) galvanometer scanhead.
  • Maximum scan format with conventional scanner 8192 x 8192 pixels
  • Scanning speeds in conventional scanning: user adjustable from 1 to 1800 Hz/lines per sec (up to 3600 Hz in bidirectional mode)
  • Scanning speed in bidirectional mode (resonant scanning): 16000 Hz/lines per sec (minimal zoom 1.3x)

Excitation sources

  • Infrared laser - Ti-Sapphire femtosecond pulsed Chameleon Ultra II (Coherent Inc.) with tuning range 680-1080 nm, 40 nm/sec tuning speed and 3.5 W average power at peak
  • Visible lasers - 405 nm (50 mW), 488 nm (20 mW), 552 nm (20 mW), 638 nm (30 mW)

Objectives

  • 25x/1.00 W motCORR HC IRAPO L, Working Distance 2.6 mm. Water immersion objective with high transmission and color correction optimized for excitation in IR up to 1300 nm. Motorized correction collar for precise and fast adjustment of the optics to changes in refractive index. Use with or without cover glass.
  • 20x/0.75 W HC PL IRAPO, WD 0.67 mm. Water immersion objective with high transmission and color correction optimized for excitation in IR up to 1300 nm. Use with or without cover glass.
  • 40x/1.1 W CORR HC PL IRAPO, WD 0.65 mm. Water immersion objective with correction collar optimized for multiphoton applications. Use with 0.14-0.18 mm cover glass.
  • 63x/0.90 W U-V-I CS2 HC APO L, WD 2.2 mm. Water immersion objective with high color correction and high transmission. Use without cover glass.
  • 63x/1.40 OIL CS2 HC PL APO, WD 0.14 mm. Oil immersion objective with superior color correction; optimized for confocal scanning applications.
  • 20x/0.95 Water IR corrected XLUMPLFL20XW, WD 2.0 mm (Olympus). For special multiphoton applications.
  • 25x/0.95 Water HCX IRAPO L, WD 2.4 mm, Cover glass 0.17 mm. For special multiphoton applications.
  • 10x/0.40 Air HC PL APO CS, WD 2.2 mm, Cover glass 0.17 mm. Air objective good for taking an overview of the sample. Not optimized for 405 nm excitation.

Detectors

  • Multiphoton mode: 4 external non descanned detector (NDD) HyD (Hybrid Detector - GaAsP with Avalanche Photodiode)
  • Confocal mode: 2 HyD detectors, 2 standard PMT (Photon Multiplier Tube) for fluorescence, 1 PMT for transmitted light

Acquisition software

  • LAS X (Leica Application Suite with SP8 control software, Dye Finder, 3D Visualization, HyVolution and Electrophysiology modules)
  • Huygens Deconvolution Package for Confocal and Multiphoton
  • Experiment/image file format: LIF (export options to TIFF, JPEG, AVI, MOV)

Accessories

  • Perfusion pump
  • Heating ring for 60 mm dishes
  • Isoflurane anesthesia unit
  • Animal temperature controller
  • Ventilator
  • Pulse Oximeter
  • Suction window for organ stabilization during intravital imaging
  • Stereomicroscope

ZEISS LSM880 AIRYSCAN FAST CONFOCAL SYSTEM

Location: Room 2113 (Microscopy Suite)

The Zeiss LSM 880 was acquired through the Shared Instrumentation Grant Program (S10) and must be cited for its use: NIH S10OD021831.

Capabilities

  • Enhanced resolution confocal imaging with Airyscan FAST detector
  • Versatile and sensitive confocal microscope
  • Confocal imaging of slides and cover glass bottom dishes
  • 3D volume and automated multi-area imaging
  • Advanced imaging with FRET and FRAP
  • Live cell imaging with environmental chamber and Definite Focus2 module

Objectives:

10x/0.45 NA EC Plan-Apochromat air objective, 2 mm working distance (WD)

20x/0.8 NA Plan-Apochromat air objective, WD 0.55 mm

40x/1.2 NA C-Apochromat water objective, with motorized correction collar, WD 0.28 mm

40x/1.4 NA Plan-Apochromat oil objective, WD 0.13 mm

63x/1.4 NA Plan-Apochromat oil objective, WD 0.14 mm, hand-selected for enhanced resolution

63x/1.46 NA alpha Plan-Apochromat oil objective, WD 0.10 mm

All objectives are capable of differential interference contrast imaging

Excitation sources:

405, 458, 488, 514, 561, 633 nm laser lines

Detectors:

Airyscan FAST GaAsP detector for enhanced sensitivity and resolution.

2 bi-alkali PMTs and a 32-channel spectral GaAsP detector.

ZEISS LSM780 CONFOCAL SYSTEM

Location: Room 2113 (Microscopy Suite)

Capabilities:

  • Versatile and sensitive inverted confocal microscope
  • Confocal imaging of slides and cover glass bottom dishes
  • 3D volume and automated multi-area imaging
  • Advanced imaging with FRET and FRAP
  • Live cell time-lapse imaging with environmental chamber

Objectives:

  • 10x/0.3 NA EC Plan-Neofluar air objective, 5.3 mm working distance (WD), DIC I
  • 20x/0.8 NA Plan-Apo air objective, 0.61 mm WD, DIC II
  • 25x/0.8 NA LD LCI Plan-Apo multi-immersion objective with correction collar for water, oil, glycerine, 0.60 WD, DIC II
  • 40x/1.3 NA EC Plan-Neofluar oil objective, WD 0.2 mm, DIC III
  • 63x/1.4 NA Plan-Apo oil objective, WD 0.19 mm, high contrast DIC
  • 100x/1.4 NA Plan-Apo oil objective, ED 0.17 mm, high contrast DIC

Excitation sources:

405, 458, 488, 514, 561, 633 nm laser lines

Detectors:

2 bi-alkali PMTs and a 32-channel spectral GaAsP detector.

ZEISS AXIOSCAN Z1 SLIDE SCANNER

Capabilities

- Automated brightfield and fluorescence imaging of whole slides.

- Barcode scanner and automated tissue detection.

- Enhanced depth of field for uneven samples. 

Location

Room 2328 (Histology Suite)

System specifications

Microscope

  • Compact self-contained unit
  • Upright configuration
  • Motorized stage and automatic focus

Illumination

Transmitted light: LED (wavelength 400 to 700 nm, maximum at 460 nm)

Fluorescence: Colibri 7 LED (385, 430, 475, 511, 555, 590, 630 nm excitation)

Fluorescence filter sets

  • High-efficiency filter set suitable for fluorescent dyes like Hoechst, AF488/GFP, AF568 and AF647 which use 385, 475, 555 and 630 nm excitation. Contains dichroic mirrors 405 + 493 + 575 + 653 and emission filters 425/30 + 514/30 + 592/30 + 709/100.
  • High-efficiency filter set suitable for fluorescent dyes like CFP, YFP and mCherry which use 430, 511 and 590 nm excitation. Contains dichroic mirrors 450 + 538 + 610 and emission filter TBP 467/24 + 555/25 + 687/145.

Air objectives

  • 10x/0.3NA
  • 20x/0.8NA
  • 40x/0.95NA

Cameras

  • Hitachi HV-F202 for brightfield (1600 x 1200 pixels)
  • Orca Flash 4.0 v2 for fluorescence (2048 x 2048 pixels, 82% peak quantum efficiency 

Acquisition software

  • ZEN2.3 slidescan module
  • Experiment/image file format: czi (export options to TIFF, JPEG, BigTIFF)
  • Images can be viewed on Windows computers with ZEN lite software (https://www.zeiss.com/microscopy/us/downloads/zen.html)

NANOIMAGER - Oxford Nanoimaging

Location:  Room 2113 (Microscopy Suite)

Capabilities

- 3D stochastic optical reconstruction (STORM) and total internal reflection fluorescence (TIRF) microscopy

- Two simultaneous imaging channels and large 50x80 micron field of view

- Autofocus, drift and vibration correction, tile stitching for overview of the imaging

- Fast, GPU based software for automated real-time localization, single-particle tracking, and single molecule fluorescence resonance energy transfer

 

System specifications

  • Compact, fully motorized inverted microscope with temperature control and advanced Z position / XY-drift stabilization
  • Laser lines: 405 nm (150 mW), 473 nm (1 W), 543 nm (1 W), and 640 nm (1 W)
  • 100x 1.49 NA Olympus TIRF objective
  • Equipped with a sensitive camera with 82% peak quantum efficiency

 

Olympus FluoView FV10i confocal microscope

Capabilities
- Confocal imaging of slides and cover glass bottom dishes
- 3D volume and automated multi-area imaging
- Automatic detection of interphase between specimen and cover glass

Location
Room 2113 (Microscopy Suite)

System specifications
Microscope

  • Compact self-contained unit
  • Inverted configuration
  • Motorized stage and focus

Scanner

  • Conventional galvanometer scanner

Excitation sources

  • 4 diode lasers (405 nm, 473 nm, 559 nm, 635 nm)

Emission ranges

  • Blue (420-520 nm)
  • Blue-Narrow (420-460 nm)
  • Green (490-590 nm)
  • Green-Narrow (490-540 nm)
  • Red (570-670 nm)
  • Red-Narrow (570-620 nm)
  • FarRed (660-760 nm)
  • FarRed-Narrow (660-710 nm)

Objectives

  • 10x/0.40 Air
  • 60x/1.35 Oil

Detectors

  • 2 standard PMT for fluorescence, 1 PMT for phase contrast

Acquisition software

  • FV10i-SW
  • Experiment/image file format: OIF (export options to TIFF, JPEG)

ZEISS Axiovert 200M Marianas deconvolution system

Capabilities
- Camera-based widefield imaging of live or fixed specimens
- Fluorescence, bright field, DIC and phase contrast microscopy
- Slides, dishes or tissue plates with cover glass bottom
- Image deblurring by nearest-neighbor deconvolution algorithm

Location
Room 2113 (Microscopy Suite)

System specifications
Microscope

  • Inverted Zeiss Axiovert 200M stand
  • DIC optics
  • Motorized stage
  • Environmental chamber for long-term imaging

Light/excitation source

  • Lambda LS Xenon arc lamp (Sutter Instruments)
  • light output 340-700 nm

Objectives

  • 5x/0.25 Air Fluar
  • 10x/0.3 Air EC Plan NeoFluar
  • 20x/0.50 Air Plan NeoFluar Ph 2 (1004-989)
  • 20x/0.50 Air Plan NeoFluar DIC (1004-072)
  • 40x/1.3 Oil Plan Neofluar
  • 63x/1.4 Oil Plan Apochromat DIC

Fluorescence filter cubes

  • Blue (DAPI, Hoechst), Excitation filter 382-392 nm, emission filter 417-477 nm
  • Green (FITC, GFP), Excitation filter 465-499 nm, emission filter 516-556 nm
  • Red (TRITC, RFP), Excitation filter 532-554 nm, emission filter 573-613 nm
  • Far Red (Cy5, APC), Excitation filter 608-648 nm, emission filter 672-712 nm

Detection

  • Photometrics’ Coolsnap HQ2 cooled CCD camera (1392 x 1040 pixel imaging array)

Acquisition software

  • SlideBook 6.0 (3i)
  • Experiment/image file format: SLD (export options to TIFF, AVI)

Nikon 80i epifluorescence microscope

Capabilities
- Widefield fluorescence imaging
- Brightfield image capture with color camera (i.e. H&E staining analysis)
- Slide viewing

Location
Room 2113 (Microscopy Suite)

System specifications
Microscope

  • Upright configuration
  • Manual stage

Excitation source

  • X-Cite 120, Mercury vapor short arc

Objectives

  • 4x/0.20 Air Plan Apo, WD 20 mm
  • 10x/0.45 Air Plan Apo, WD 4.0 mm, Cover glass 0.17 mm
  • 20x/0.75 Air Plan Apo, WD 1.0 mm, DIC, Cover glass 0.17 mm
  • 40x/0.95 Air Plan Apo, WD 0.14 mm, DIC Prism; Selectable cover glass 0.11-0.23 mm
  • 60x/1.4 Oil Plan Apo VC, WD 0.13 mm, Cover glass 0.17 mm
  • 100x/1.25 Oil Plan Apo, WD 0.17 mm, Cover glass 0.17 mm

Fluorescence filter cubes

  • Blue (DAPI, Hoechst), Excitation filter 340-380 nm, dichroic mirror 400 nm, emission filter 435-485 nm
  • Green (FITC, GFP), Excitation filter 465-495 nm, dichroic mirror 505 nm, emission filter 515-555 nm
  • Red (TRITC, RFP), Excitation filter 540-580 nm, dichroic mirror 595 nm, emission filter 600-660 nm
  • Spectral, Excitation filters: 400-414 nm, 484-504 nm, 566-586 nm, transmission bands: 446-468 nm, 520-540 nm, 614-642 nm, emission filters: 446-468 nm, 520-540 nm, 614-642 nm.

Detection

  • Leica DFC450 digital camera

Acquisition software

  • LAS AF (Leica Application Suite Advanced Fluorescence)
  • Experiment/image file format: Leica Image Format - LIF, export to TIFF, JPEG

Imaging Workstations

Capabilities
- Three high-end imaging workstations for data analysis and visualization
- Wide range of image and data processing software installed

Location
Room 2113 (Microscopy Suite)

System specifications
Software

  • Imaris 9.1 (two floating licenses)
  • Image-Pro Premier 9.2
  • Olympus FV10-ASW
  • Zeiss ZEN2.3
  • Leica LASX
  • SlideBook 6.0
  • XuvStitch 1.8.1
  • ImageJ/Fiji
  • Matlab
  • Adobe Illustrator CS6
  • Adobe Photoshop CS6
  • Adobe Premier Pro CC 2017
  • Canvas 14
  • GraphPad Prism 7

Workstation I

  • Windows 7 Professional
  • Intel Xenon X5570 2.93 GHz (8 cores)
  • 24 GB RAM memory
  • ATI FirePro V8700 with 1 GB memory
  • 0.5 TB SSD system drive and 1 TB HDD local storage

Workstation II

  • Windows 7 Professional
  • Intel Xenon E5-2689W v2 3.40 GHz (16 cores)
  • 64 GB RAM memory
  • AMD Radeon R9 200 with 4 GB memory
  • 0.5 TB SSD system drive and 2 TB HDD local storage

Workstation III

  • Windows 7 Professional
  • Intel Xeon CPU E5-1650 v3 3.5GHz (12 cores)
  • 128 GB RAM memory
  • NVIDIA Quadro M4000 with 8 GB memory
  • 0.5 TB SSD system drive, 2 TB SSD for processing and 4 TB HDD local storage

Each workstation is equipped with two 32” Dell U3014 color calibrated monitors and connected to LJI servers for secure data storage.

Histology instrumentation

  • The following histology equipment is available to trained users:

    • Automated Tissue Processor – Sakura VIP150
    • Embedding Station – Shandon Histocentre 2
    • Microtome – Leica RM2125 RTS
    • Tissue Flotation Bath – Boekel 145702
    • Hybridization Oven – Hybad
    • Vibratome – Vibratome 1000 Plus
    • Cryostat Microtome – Leica CM 1850
    • Pressure cooker for antigen retrieval
    • Sequenza slide racks for more efficient immunostaining
    • Light microscope

Images

Rates

  2018 Internal Rates 2018 External Rates
Histology Core Services
Block with one HE slide (This includes cassette of tissue, processing, embedding, cutting, and staining with HE) $7.00 $13.23
Block Only (Includes all of the above except cutting) $3.00 $5.67
Block with one (1) unstained slide (Includes all of the above except for the staining of the cut slide) $4.25 $8.03
Extra unstained slide $1.25 $2.36
Extra HE slide $2.75 $5.20
Special Stain II (Stains for carbohydrates, connective tissue, muscle fibers, lipids, pigments, minerals, and nerve tissue per slide) $8.00 $15.12
Special Stain III (Stains for microorganism, special cells using silver impregnation) $9.00 $17.01
Frozen Sectioning $3.25 $6.14
Assay handling, special project (per hour) $30.00 $56.70
Decal $1.00 $1.89
Consultation/Training (per hour) $70.00 $132.30
Microscopy Support Services
Leica SP8 (confocal/multiphoton imaging) $30.00 $56.70
Leica SP8 after hours 10pm-8am (confocal/multiphoton imaging) $15.00 $28.35
Leica SP8 (confocal/multiphoton imaging), assisted $80.00 $151.20
Olympus FV10i (confocal) $10.00 $18.90
Olympus FV10i (confocal), assisted $60.00 $113.40
Nikon 80i (widefield, fluorescence and H&E staining) $5.00 $9.45
Nikon 80i (widefield, fluorescence and H&E staining), assisted $55.00 $103.95
Marianas Zeiss Axiovert 200M (widefield, fluorescence and DIC) $10.00 $18.90
Marianas Zeiss Axiovert 200M (widefield, fluorescence and DIC), assisted $60.00 $113.40
IVIS (in vivo luminescence and fluorescence detection system) $5.00 $9.45
IVIS (in vivo luminescence and fluorescence detection system), assisted $55.00 $103.95
Imaging workstation (Imaris, ImagePro, Slidebook) $5.00 $9.45
Imaging workstation (Imaris, ImagePro, Slidebook), assisted $55.00 $103.95
AxioZ1 slide scanner $30.00 $56.70
AxioZ1 slide scanner, assisted $80.00 $151.20
Zeiss LSM780 confocal $20.00 $37.80
Zeiss LSM780 after hours, 10 pm - 8 am) $10.00 $18.90
Zeiss LSM780 confocal (assisted) $70.00 $132.30
LSM 880 with Airyscan (unassisted, per hour) $25.00 $47.25
Nanoimager (per hour) $35.00 $66.15
Microscopy Training Services
Training and Consultation (per hour) $70.00 $132.30
Assisted Usage (per hour) $50.00 $94.50
3D Printing
Material Printing $0.50/hr + $50/kg $0.95/hr +$94.50/kg
Training/hour $70.00 $132.30
Project Support/Design (per hour) $55.00 $103.95
Design Software (per hour) $5.00 $9.45

Resources

Histology Resources on the Web

The Internet Pathology Laboratory

Histomorphologic Evaluation of Lymphoid Organs