Nikon TIRF/HiLo

This microscope combines a total internal fluorescence (TIRF) and highly inclined and laminated optical sheet (HiLo: https://www.nature.com/articles/nmeth1171) modalities in one instrument. The TIRF modality allows user to image molecules at surface of cells and apply super- resolution imaging techniques such as PALM and STORM. HiLo modality offers the possibility of exciting a thin inclined plane within cells or thin layer of cells within few micrometers of a live animal tissue. Automatized variability of angle of HiLo emergence and camera imaging allows for fast sectioning of sample. The system is also supplemented with NIDAQ board and control with external trigger, which combined with the versatile illumination sequence option makes this ideal microscope for complex experiments involving the correct timing of imaging following an external stimulus of cells or animal. Definite focus is perfectly controlled using Nikon PFS system making it ideal for long term live cell or animal imaging.

Microscope body:	Inverted
Motorised Stage:	Yes
Temp Control:	Fully Enclosed
CO₂:	Stage Insert
Software:	NIS Elements & Jobs

Common Applications

Live Cell Imaging:	Many biological processes are dynamic events and as such observing living cells is crucial to unravelling and understanding the mechanisms behind them. This requires special considerations when imaging. Firstly, the environmental conditions must be controllable and stable to ensure the cells remain healthy. Secondly care needs to be taken that the act of imaging has as little impact as possible on the event being observed. This microscope has fully enclosed heated chamber and CO2 supply for short or long term live cell imaging.
Cell Migration:	This broad term is used to refer to any processes that involve the translation of cells from one location to another. This may include development, wound healing, invasion of cancer cells, immune response. This microscope is equipped with definite focus system, PFS, which is perfect for this type of application.
TIRF	Total Internal Reflection Fluorescence microscopy exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices. In practice, the most commonly utilized interface in the application of TIRF is the contact area between a cell and a glass coverslip or tissue culture container.
Single Molecule Imaging	Single molecule at the surface of a cell using TIRF (N-STORM) modality or within single cross section of cells using HiLo modality. Cooled EMCCD camera is perfect for capturing single molecules at detectable signal to noise.
Intravital Imaging	Intravital imaging is the observation of biological processes in living animals/tissue. It introduces new challenges such as achieving higher penetration depths and dealing with sample mobility. Appreciating that how cells respond in tissue culture has often been shown to be largely different from their response in their true environment makes intravital imaging a crucial step in understanding biological systems. This microscope can do intravital imaging at single molecule level within first few micrometers of an epithelium.

Objective Lenses

MAG	N.A	CORRECTIONS	IMMERSION	W.D	MISC.
20x	0.75	Plan Apochromat	AIR	1 mm	DIC/Pol
60x	1.49	Apochromat	OIL	0.12 mm	DIC/Pol
100x	1.49	Apochromat	OIL	0.12 mm	DIC/Pol

Note: All lenses are compatible with definite focus PFS. 20 X lens is suitable for dark field microscopy. 60 and 100 x can be used with UV illumination but not recommended.

Light Sources

Source	Wavelengths	Output Power
Laser OBIS 405	405 nm	100 mW
Laser OBIS 488	488 nm	100 mW
Laser OBIS 561	561 nm	100 mW
Laser OBIS 640	640 nm	100 mW
Mercury Lamp	400-600 nm

Filters

NAME	EXCITATION	DICHROIC	EMISSION
DAPI WF	BP 340 - 380 nm	LP 400 nm	BP 435 - 485 nm
GFP WF	BP 465 - 495 nm	LP 505 nm	BP 515 - 555 nm
TxRd WF	BP 528 - 553 nm	LP 565 nm	BP 590 - 650 nm

NAME	EXCITATION	DICHROIC	EMISSION
Cy3/Cy5	BP 572 - 621 nm	LP 600 nm	-
FITC/TRITC	BP 470 - 490 nm BP 540 - 565 nm	-	BP 503- 530 nm BP 580 - 620 nm
FITC	BP 465 - 595 nm	LP 505 nm	BP 515- 555 nm

NAME

EXCITATION

DICHROIC

EMISSION

Cy3/Cy5

BP 572 - 621 nm

LP 600 nm

FITC/TRITC

BP 470 - 490 nm

BP 540 - 565 nm

BP 503- 530 nm

BP 580 - 620 nm

FITC

BP 465 - 595 nm

LP 505 nm

BP 515- 555 nm

Single Band (405, 488, 561/568, 635 nm) BrightLine and dual (488 +561 nm) BrightLine Sedat laser blocking filters.

Detector

Model	Pixel format	Sensor Type	Readout Speed (MHz)	Description
Andor 897 Ultra	512x512 (16um pixel size)	EMCCD	1, 5, 10 and 17	Sensitivity and speed at 56 fps at full 512x512 or 595 fps with 128 x 128 crop mode

Common Applications

Objective Lenses

Light Sources

Filters

Detector

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