Fluorescence-guided stereotactic biopsy: a proof-of-concept study

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Accurate histopathological diagnoses are often necessary for treating neuro-oncology patients. However, stereotactic biopsy (STB), a common method for obtaining suspicious tissue from deep or eloquent brain regions, fails to yield diagnostic tissue in some cases. Failure to obtain diagnostic tissue can delay initiation of treatment and may result in further invasive procedures for patients. In this study, the authors sought to determine if the coupling of in vivo optical imaging with an STB system is an effective method for identification of diagnostic tissue at the time of biopsy.


A minimally invasive fiber optic imaging system was developed by coupling a 0.65-mm-diameter coherent fiber optic fluorescence microendoscope to an STB system. Human U251 glioma cells were transduced for stable expression of blue fluorescent protein (BFP) to produce U251-BFP cells that were utilized for in vitro and in vivo experiments. In vitro, blue fluorescence was confirmed, and tumor cell delineation by fluorescein sodium (FNa) was quantified with fluorescence microscopy. In vivo, transgenic athymic rats implanted with U251-BFP cells (n = 4) were utilized for experiments. Five weeks postimplantation, the rats received 5–10 mg/kg intravenous FNa and underwent craniotomies overlying the tumor implantation site and contralateral normal brain. A clinical STB needle containing our 0.65-mm imaging fiber was passed through each craniotomy and images were collected. Fluorescence images from regions of interest ipsilateral and contralateral to tumor implantation were obtained and quantified.


Live-cell fluorescence imaging confirmed blue fluorescence from transduced tumor cells and revealed a strong correlation between tumor cells quantified by blue fluorescence and FNa contrast (R2 = 0.91, p < 0.001). Normalized to background, in vivo FNa-mediated fluorescence intensity was significantly greater from tumor regions, verified by blue fluorescence, compared to contralateral brain in all animals (301.7 ± 34.18 relative fluorescence units, p < 0.001). Fluorescence intensity measured from the tumor margin was not significantly greater than that from normal brain (p = 0.89). Biopsies obtained from regions of strong fluorescein contrast were histologically consistent with tumor.


The authors found that in vivo fluorescence imaging with an STB needle containing a submillimeter-diameter fiber optic fluorescence microendoscope provided direct visualization of neoplastic tissue in an animal brain tumor model prior to biopsy. These results were confirmed in vivo with positive control cells and by post hoc histological assessment. In vivo fluorescence guidance may improve the diagnostic yield of stereotactic biopsies.

ABBREVIATIONS BFP = blue fluorescent protein; FNa = fluorescein sodium; RFU = relative fluorescence units; ROI = region of interest; STB = stereotactic biopsy.

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Article Information

Correspondence Joseph Georges: Philadelphia College of Osteopathic Medicine, Philadelphia, PA. joseph.georges@asu.edu.

INCLUDE WHEN CITING Published online February 22, 2019; DOI: 10.3171/2018.11.JNS18629.

R.L., M.I., and J.G. contributed equally to this work.

Disclosures The authors report no conflict of interest concerning the mdaterials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.



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    Biopsy needle and microendoscope. A: Labeled illustration of imaging system with microscope base, fiber optic microendoscope, rodent with implanted xenograft positioned with stereotactic apparatus, and image display. B: Biopsy needle outer cannula modified with distal opening (left), biopsy needle inner cannula (middle), and 0.65-mm-diameter fiber optic microendoscope (right). C: Biopsy needle with distal end removed and inner cannula (circle) inserted. D: Outer cannula of biopsy needle with inner cannula removed and microendoscope inserted (arrow). Bar units in millimeters.

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    Molecular imaging of fluorescent glioma cells with fluorescein contrast. A: Confocal microscopy confirms expression of BFP in modified human glioma cells. B: Fluorescein contrast delineates glioma cell bodies. C: Merged image of panels A and B. D: Coefficient of correlation between BFP-expressing cells and cells delineated with FNa-mediated fluorescence contrast. Pearson (R2) = 0.91 with 95% confidence intervals (dotted lines) of 0.90–0.98, p < 0.001. Bar = 20 µm.

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    Fluorescein differentiates tumor from normal brain in vivo. Tumor (A–C). BFP-expressing tumor (A), corresponding FNa-mediated fluorescence image (B) and merged image (C). Normal brain (D–F). Absence of blue fluorescence (D), minimal corresponding fluorescein signal (E), and merged image (F). H & E staining of biopsy specimen from a region of strong fluorescein signal (G) shows hypercellular tumor compared to contralateral brain with minimal fluorescein signal (H). In vivo fluorescence endomicroscopy (I) shows significantly greater FNa-mediated fluorescence intensity from tumor regions compared to normal brain across animal models, and mean fluorescence from tumor regions (J) is 301.7 ± 34.18 RFU greater than background fluorescence (p < 0.001). Bar = 20 µm.




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