23% of cells are PR+ (> OD of 0.1)
10% of cells are ER+ (> OD of 0.1) Two cell phenotypes detected.
ER – stained serial section (SG-green)
Patho 2011-8-12
多光谱成像及定量分析系统在 病理诊断中的初步应用
Patho 2011-8-12

CRI多光谱显微成像系统
Patho 2011-8-12
什么是多光谱成像?
Nuance 基于液晶可调谐滤光技术，可以在不同波长处检测荧光探针或染料在细胞或组织中的分布
YFP signal
420nm 490nm 720nm
前列腺组织特异性 膜抗原
RGB
525 nm (20-nm bandpass)
探针: 525-nm QDs
Unmixed
Composite image
MSI技术：显著提高了组织切片的信噪比
Patho 2011-8-12
CD147+p53
多光谱分离技术：揭示更多的分子靶点标记
Patho 2011-8-12
PR
ER
Hematoxylin
Patho 2011-8-12
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Sample courtesy DAKO
Serial sections can generate two independent cell phenotypes from two stains
PR – stained serial section (VIP-purple)
Multiplexing can generate multiple co-expression cell phenotypes from two stains
ER/PR – double-stained section (ER-SG, PR-VIP)
PR+ / ER-
15%
PR- / ER-
81%
CISH performed using c-Myc translocation for t(8;14) on Burkitt’s lymphoma sectioned cell pellet
Patho 2011-8-12
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Punctate signals were related to progression only in basal cells, identifying CIN1 with potentially aggressive behavior. GPyantehcoo2l0o11g-i8c-1O2 ncology 112 (2009) 114–118
种类:
• •
特征:
• • • • •
420 to 720 nm 500 to 950 nm
电子可调谐带宽滤光片 获得随机波长带宽 优质的光学质量 – 固定像素点, 无可移动部件 快速的波长调节 优质的光谱分离
LCTF (randomly tunable) CovePreadthboy 2pa0t1e1n-t8s-51,2953,087, 4,848,877, 5,247,378 and patents pending
DAPII + KKii6677 + CD3 + CCDD2200++IIggDD+ CD68
Patho 2011-8-12
非典型性导管增生的荧光原位杂交
Sample Fluorophores: • Chromosome 17 (FITC) • Her2 (Cy3) • Nuclei (DAPI) • Tissue (Autofluorescence)
Patho 2011-8-12
临床相关研究，抗原共定位的分离：乳腺癌ER和PR
RGB image (from spectral stack)
Progesterone receptor: DAB Estrogen receptor: Fast Red Counterstain: hematoxylin
Unmix: • DAPI signal into blue channel • FITC signal into the green channel • Cy3 signal into the red channel • Autofluorescence into “black”
Patho 2011-8-12
* Courtesy Dan Pinkel and Donna Albertson, UCSF
双色素原位杂交法检测Burkitt淋巴肿瘤细胞株
Unmixed into simulated fluorescence mode
Red = chrom. 8 Green = chrom. 14
Yellow or adjacent red and green = 8:14 translocation
Monochrome
Color (RGB)
多光谱分离技术：显著提高了细胞成像的信噪比
Patho 2011-8-12
Multispectral
多光谱的原理
光谱vs颜色
两种黄色无法区分
Patho 2011-8-12
光谱存在明显差异
基于液晶可调谐滤光片 (LCTF)的多光谱成像技术
Lyot filter (fixed bandpass)
PR+ / ER+
4%
PR- / ER+
0%
Detects 4 cell phenotypes, not just two.
多光谱技术的实现:LCTF+软件系统-发射光光谱曲线
1. CCD采集不同波长的图像 2. 数据转化成一个 “cube”
565nm
5 量子点探针
655nm
3. CCD每个像素均得到光谱曲线
650 nm
625 nm
z
x
600 nm
575 nm
550 nm
y
Patho 2011-8-12
Spectra
多光谱分离：完全去除组织切片的强自发荧光
Her-2+CK 乳腺癌
Patho 2011-8-12
多光谱分离技术:多色IHC标记的检测与分离
RGB
Spectra of 5 Qdots
Unmixed composite 淋巴细胞数量分化过程中，对淋巴 生发中心的标记： 5 种量子点比较 DAPI 细胞核标记
Patho 2011-8-12
多光谱分离技术:多信息数据挖掘