Imaging

Originally sponsored by the James P. Wilmont Cancer Center, the acquisition and upgrading of the Amnis ImageStream was supported by NIH Grant 1 S10 RR023458-01A1 (C Jordan PI).

Amnis - ImageStream

Fee Structure
Guidelines
Configuration - Brightfield || Darkfield || 488 Laser || 405 Laser || 658 Laser
Fluorochrome Chart

Amnis ImageStream, a hybrid fluorescent microscope/flow cytometer. With this technology, one can collect morphological data on cells stained with up to four different fluorochromes at 5,000 events per minute. Coupled with the analytical packages, IDEAS, statistical rigor can be brought to answer questions such as:

sub-cellular protein localization
surface maker co-localization
marker asymmetry
molecular trafficking
Phagocytosis ll

ImageStream

Fee Structure

$50/hour

Guidelines - Coming Soon

Configurations

Brightfield - Each channel has a certain bandpass limit listed below.

	Channel 1	Channel 2	Channel 3	Channel 4	Channel 5	Channel 6
BP	OPEN	450/40	530/25	577/10	630/10	694/11

Darkfield - Each channel will resolve dyes of certain wavelengths as listed below.

	Channel 1	Channel 2	Channel 3	Channel 4	Channel 5	Channel 6
BP	470-500	400-470	500-560	560-595	595-660	660-735

Based on the above tables, the laser configuration below can excite the listed dyes. After all three lasers, a table is shown that gives a visual representation of which channel each excited dye will be resolved.

488 Laser (Alphabetical by Channel)

Channel	Channel 3	Channel 4	Channel 5	Channel 6
Fluor's	AF 488	AF 546	7AAD	Alexa 647
	Fluorescein	AF 555	Alexa 610	Alexa 680
	GFP	Cy-3	ECD	Draq5
	Spectrum Green	DSRed	PI	PE-Cy5
	Sybr Green	PE	PE-Alexa 610	PE-Cy5.5
	Syto Green		PE-Texas Red	PE-Alexa 647
	YFP			PE-Alexa 680
				PerCP
				PerCP-Cy5.5
				QD-705

405 Laser (Alphabetical by Channel)

Channel	Channel 2	Channel 3
Fluor's	AF 405	Pacific Orange
	AF 430
	Cascade Blue
	DAPI
	Hoechst
	Marina Blue
	Pacific Blue

658 Laser (Alphabetical by Channel)

Channel	Channel 6
Fluor's	AF 647
	AF 680
	APC
	Cy5
	Cy5.5

Channel 1	Channel 2	Channel 3	Channel 4	Channel 5	Channel 6
470-500	400-470	500-560	560-595	595-660	660-735
Darkfield	Brightfield	Brightfield	Brightfield	Brightfield	Brightfield
	DAPI	Fluorescein	PE	7AAD	PE-Cy5
	Hoechst	AF 488	Cy-3	Alexa610/PE	PE-Cy5.5
	Pacific Blue	GFP	AF 546	PI	Alexa680/PE
	Cascade Blue	Syto Green	AF 555	PE-Texas Red	Alexa647/PE
	Marina Blue	Spectrum Green	DSRed	ECD	PerCP
	AF 430	YFP			PerCP-Cy5.5
	AF 405	Sybr Green			Draq-5
		Pacific Orange			QD-705
					APC
					AF680
					AF647
					Cy5
					Cy5.5

Fluorchromes listed in black are excited by the 488nm laser, those listed in blue by the 405nm laser, and those in red by the 658nm laser.

ImageStream GenX

Fee Structure

$50/hour

Guidelines - Coming Soon

Configurations

Channel Specifics

	Channel 1	Channel 2	Channel 3	Channel 4	Channel 5	Channel 6
Upper End	505	560	595	660	740	800
Peak	467.5	532.5	577.5	627.5	700	770
BP +/-	37.5	27.5	17.5	32.5	40	30
	Channel 7	Channel 8	Channel 9	Channel 10	Channel 11	Channel 12
Upper End	505	560	595	660	740	800
Peak	467.5	532.5	577.5	627.5	700	770
BP +/-	37.5	27.5	17.5	32.5	40	30

Based on the above table, the laser configuration below can excite the listed dyes. After each laser, a table is shown that gives a visual representation of which channel each excited dye will be resolved.

Full Fluorchrome List

Use the following link to access the full list of fluors usable on this instrument. If a fluor you would like to use is not listed, contact Dave Fuller in the Flow Core.

Flurochrome List

Upcoming Events

Wednesday at 10am, MRB 3-9654

10/14 Multichromatic Panel Design: Practical

10/28 An Introduction to Fluorescence Microscopy

11/3 - Tuesday at 11AM Introducing the Guava 8HT personal flow cytometer

Training Lecture - MC 3-6232

Sept 28, Oct 12, Oct 26