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HOME> Products> Miscellaneous> HistoBright
HOME> Products> Cell Culture> Imaging dye> HistoBright

Tissue Structure Visualizing Fluorescent Dye HistoBright

Date：December 01 2023Web Page No：520068

Funakoshi Co.,Ltd.

HistoBright is an environment-responsive membrane-staining fluorescent dye which can be exited by the two photon laser. It has high tissue permeability, and its fluorescent properties change depending on the local structure of the tissue, allowing high-contrast tissue visualization. Combining tissue clearing techniques with two photon laser microscopy allows deep-imaging and spatial structural analysis of tissue blocks.

HistoBright is an environment-responsive fluorescent dye (solvatochromic dye) that exhibits a wide range of wavelengths depending on the polarity of the solvent. According to this property, the fluorescent colors of HistoBright change in response to microenvironment within the tissue. Combining tissue clearing methods with two photon laser microscopy clearly visualized 3D tissue structure.

Historical Colorimetric Methods & Novel Fluorescent Method for Tissue Structure Analysis
Features
Original Paper
Reference Data
Application Data
Product Information

Historical Colorimetric Methods & Novel Fluorescent Method for Tissue Structure Analysis

Tissue structures are historically visualized by staining with various colorimetric dyes such as Hematoxylin and Eosin (HE) stains. These conventional colorimetric stains generally require thinly sliced tissue sections and are unsuitable for thick tissue blocks. However, the preparation of thinly sliced tissues loses the 3D structural information. To reconstitute 3D information, a lot of additional thin slices and stainings are required.

HistoBright (original compound name; PC in original paper) is originally synthesized by Dr. Yosuke Niko, Kochi University, and Dr. Takeshi Imamura, Dr. Masamoto Murakami, and Dr. Ryosuke Kawakami, Ehime University, promotes its biological 3D imaging application. HistoBright is a novel solvatochromic fluorescent dye that changes fluorescent colors in response to solvents or microenvironments and has a high affinity to various biological membranes. The combination of two features of HistoBright contributes to clear and bright imaging of tissue structures. As HistoBright can be excited by not only one-photon but also two-photon, HistoBright is compatible with two-photon microscopy and useful for tissue imaging. Furthermore, HistoBright, coupled with several tissue-clearing methods such as LUCID and RapiClear, which do not require lipid removal, contributes to analyzing 3D deep tissue imaging of thick tissue slices or blocks, etc., without breaking pathophysiological tissue structures. HistoBright is an innovative fluorescent dye for the visualization of 3D tissue structures.

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Features

HistoBright is an environment-responsive membrane staining reagent. Since the fluorescence characteristics change from green to near-infrared depending on the surrounding environment, local structures of tissues can be visualized in high contrast by separating the wavelength range of fluorescence to be detected.
Combined with a tissue clearing methods such as RapiClear, LUCID, which does not involve lipid removal steps, a three-dimensional analysis of large tissue structures is possible.
Both confocal microscopy (recommended excitation laser: 488 nm laser) and two photon microscopy (recommended laser: 960 nm laser) are compatible.
In the case of two photon laser microscopy, SHG (second harmonic generation) imaging can be compatible with HistoBright and collagen fibers can be simultaneously visualized without any additional staining.
After fluorescent observation of tissue with HistoBright, tissue can be used for conventional HE staining.

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Original Paper

Inoue, K., et al., J. Mater. Chem. B., 10(10), 1641～1649 (2022). Synthesis and photophysical properties of a new push-pull pyrene dye with green-to-far-red emission and its application to human cellular and skin tissue imaging. [PMID：35194628]

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Reference Data

Solvatochromic Property of HistoBright

Absorbance and fluorescent spectrum of HistoBright in various solvents. While the absorbance spectrum of HistoBright is not sensitive to solvent polarity, HistoBright changes fluorescent color from green to far-red depending on solvent polarity. Under a low polarity environment, HistoBright emits green color. On the other hand, under a high polarity environment, HistoBright emits red to far-red with low quantum yield.

Solvent	Ex_max (nm)	Em_max (nm)	Quantum yield Φ
Toluene	424	546	0.82
EtOAc	418	581	0.77
CHCl₃	427	610	0.83
MeCN	414	641	0.65
EtOH	427	691	0.2
MeOH	426	712	0.07

Fluorescent Spectrum on Liposome Membrane Model

HistoBright Fluorescent spectrum change in liposome membrane model

HistoBright rapidly accumulates in lipid membranes and emits different fluorescent colors in response to the membrane environments. On the liquid order (Lo) model liposome (sphingomyelin (SM)/cholesterol (Chol)), HistoBright emits a maximum of around 575 nm fluorescence with a high quantum yield value (0.72). On the other hand, HistoBright on the liquid disorder (Ld) model liposome (DOPC) exhibits significantly broad and relatively weak fluorescence (quantum yield 0.37), and the maximum wavelength is red-shifted by approximately 30 nm compared to the Lo model.

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Application Data

Two-Photon Fluorescence Microscopy Imaging of Human Normal Skin Tissue

After fixation of human skin tissue blocks with 4% paraformaldehyde/PBS, 500 μm thick slides were prepared and treated with the LUCID clearing reagent and 10 μM HistoBright for 76 hours. The tissue slides were observed by two-photon fluorescence microscopy (Ex. 960 nm, Em. cyan channel: 492 nm as SHG, green channel: 500-550 nm, orange channel: 560-593 nm, and red channel: 593-690 nm). The merged image was obtained from four channels.
※SHG (492 nm) is a signal derived from collagen fibers, not from HistoBright.

3D images were reconstructed from Z-stack sequences (step size, 5 μm) of 2D images.

3D image: human skin tissue by two-photon fluorescence microscopy

Two-Photon Z-stack Imaging of Human Normal Skin Tissues

After fixation of human skin tissue blocks with 4% paraformaldehyde/PBS, 1 mm thick slides were prepared and treated with the RapiClear clearing reagent and 10 μM HistoBright and Hoechst 33342 for 76 hours. The tissue slides were observed by two-photon fluorescence microscopy (Ex. 1,100 nm, Em. cyan channel: 492 nm, green channel: 500-550 nm, orange channel: 560-593 nm, and red channel: 593-690 nm). The merged image was obtained from four channels. Continuous movie data were constructed from Z-stack sequences (step size, 5 μm) of 2D images.

Dermis Imaging

Epidermis Imaging

HE Staining after HistoBright Fluorescent Imaging

After fluorescent imaging, the stained tissue block was washed with PBS. Thin tissue slices were prepared from the tissue block and re-stained with the conventional HE staining procedure.

Comparison between Confocal Microscopy and Two-Photon Microscopy Imaging

After fixation of human skin tissue blocks with 4% paraformaldehyde/PBS, 500 μm thick slides were prepared and treated with the RapiClear clearing reagent and 10 μM HistoBright for 72 hours. Tissues were observed by confocal microscopy (488 nm laser) and twophoton microscopy (960 nm laser).

Human dermal tissue by confocal microscopy & two-photon microscopy

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Product Information

[Date : April 25 2025 00:06]

Detail Product Name Product Code Supplier Size Price

HistoBright, Tissue Structure Fluorescent Dye
DatasheetThis may not be the latest data sheet. FDV-0051 FNAFunakoshi Co.,Ltd. 0.1 mg $450

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Description	HistoBright is a novel solvatochromic fluorescent dye that changes fluorescent colors in response to solvents or microenvironments and has a high affinity to various biological membranes. The combination of two features of HistoBright contributes to clear and bright imaging of tissue structures.
Storage	-20°C	CAS
Link

[Date : April 25 2025 00:06]

HistoBright, Tissue Structure Fluorescent Dye

DatasheetThis may not be the latest data sheet.

Product Code: FDV-0051
Supplier: FNA
Size: 0.1mg
Price: $450

Description	HistoBright is a novel solvatochromic fluorescent dye that changes fluorescent colors in response to solvents or microenvironments and has a high affinity to various biological membranes. The combination of two features of HistoBright contributes to clear and bright imaging of tissue structures.
Storage	-20°C	CAS
Link

Contact

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