SLICE-compatible fluorescent dyes and proteins

SLICE excitation and emission filter wavelengths

SLICE is a light-sheet microscope designed for standardized, multicolor imaging of cleared tissue. Achieving optimal performance, however, requires careful selection of fluorescent dyes tailored to the system's excitation laser and emission filters. Here we outline fluorescence channel configurations and recommend compatible dyes to help you obtain the highest signal-to-background ratio and photostability for extended imaging sessions. SLICE supports three-color imaging using the following excitation and emission channels:

	Excitation	Emission filter
Blue	450 nm	513±11 nm
Green	520 nm	576±12 nm
Red	640 nm	680±15 nm

Note that brain tissue is known to exhibit autofluorescence in the blue channel due to the presence of melanin, vitamin A1 derivatives, and lipofuscin. This can significantly reduce the signal-to-background ratio in the blue channel. Consider employing an autofluorescence quenching strategy during sample preparation to mitigate background fluorescence.

Fluorophore Choices

Organic Dyes

Organic dyes are generally preferred for imaging with the SLICE system due to their superior brightness, photostability, and resistance to photobleaching.

* Recommended dyes for SLICE imaging

Channel	Dye	Excitation Efficiency	Emission Efficiency
Blue	Alexa 430	88%	14%
	Alexa 488	14%	36%
	Atto 465	99%	26%
	iFluor 450	99%	34%
	Cyan 500	100%	26%
	YOYO-1	36%	33%
	SYTOX blue	91%	15%
	Oregon Green	17%	26%
	FITC	46%	43%
	Brilliant Violet 480	91%	21%
	Pacific Green	49%	27%
Green	Alexa 514	99%	18%
	Alexa 532	65%	24%
	Alexa 546	31%	44%
	Alexa 555*	51%	34%
	Cy3	51%	42%
	PE-Texas Red	55%	7%
	SYTOX green	33%	22%
Red	Alexa 633	83%	22.5%
	Alexa 635	86%	18%
	Alexa 647*	83%	46%
	Alexa 660	77%	45.4%
	Cy5	84%	47%
	eFluor 660	77%	47%

Fluorescent Proteins

While fluorescent proteins offer genetic specificity, their brightness and photostability often fall short of organic dyes.

		Excitation Efficiency	Emission Efficiency
Blue	GFP	49%	44%
	NeuroTrace 500/525	27%	23%
	CFP Blue	97%	22%
	eGFP	47%	43%
	mTurquoise2	92%	19%
	mCerulean3	93%	19%
	mNeonGreen	14%	44%
Green	tdTomato	60%	28%
	RFP	41%	23%
	FusionRed	26%	5%
	Citrine	87%	14%
	eYFP	77%	15%
	mCherry	26%	13%
Red	APC	79%	30%