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. 2023 Mar 7;13(6):961.
doi: 10.3390/nano13060961.

Clot Imaging Using Photostable Nanodiamond

Samuel J Francis  1 , Marco D Torelli  2 , Nicholas A Nunn  2 , Gowthami M Arepally  1 , Olga A Shenderova  2
Affiliations

Clot Imaging Using Photostable Nanodiamond

Samuel J Francis et al. Nanomaterials (Basel). .

Abstract

While thrombosis is the leading cause of morbidity and mortality in the United States, an understanding of its triggers, progression, and response to anticoagulant therapy is lacking. Intravital fluorescence microscopy has advanced the study of thrombus formation by providing targeted, multi-color contrast. However, photodegradation of fluorophores limits the application in longitudinal studies (e.g., clot progression and/or dissolution). Fluorescent nanodiamond (FND) is a fluorophore which utilizes intrinsic fluorescence of chromogenic centers within and protected by the diamond crystalline lattice. Recent developments in diamond processing have allowed for the controlled production of nanodiamonds emitting in green or red. Here, the use of FND to label blood clots and/or clot lysis is demonstrated and compared to commonly used organic fluorophores. Model ex vivo clots were formed with incorporated labeled fibrinogen to allow imaging. FND was shown to match the morphology of organic fluorophore labels absent of photobleaching over time. The addition of tissue plasminogen activator (tPa) allowed visualization of the clot lysis stage, which is vital to studies of both DVT and pulmonary embolism resolution.

Keywords: blood; fluorescence; imaging; microscopy; nanodiamond; nanoparticles; thrombus.

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Conflict of interest statement

M.D.T. and O.A.S. are employees of Adamas Nanotechnologies, Inc. which manufactures nanodiamonds. No other conflicts exist.

Figures

Figure 1
Figure 1
(A) Bulk appearance of a clot after ex vivo development in a serum separator tube and subsequent washing. Clots formed are several millimeters in size. (B) Colorized scanning electron microscopy (SEM) image of a clot demonstrating the fibrin lattice (arrow), erythrocytes (asterisk), and platelets (circle).
Figure 2
Figure 2
Views of a clot under various conditions. (A) Clot alone, with erythrocytes circled and arrowed; (B) clot + FND (green) alone (no biotinylated fibrinogen), with individual particles highlighted with arrows; (C) clot + fibrinogen FITC; (D) clot + biotinylated fibrinogen + avidin-FND (green); (E) clot + biotinylated fibrinogen + avidin-FND (red); and (F) excitation and emission spectra for NV and NVN containing FND. Scale bars 50 µm. Green Ex/Em: 470/488LP. Red Ex/Em: 517/561LP.
Figure 3
Figure 3
Avidin FND (red) containing NV centers and biotinylated fibrinogen viewed at 100× under blue excitation in formed clots. (A) 60 nm FND, (B) 120 nm FND. Scale bar 50 µm. Ex/Em: 517/561LP.
Figure 4
Figure 4
Epifluorescence microscopy examining fibrinogen labeled with FITC or FND in clot 1 h after formation before and after exposure to tPA: Clot containing fibrinogen-FITC without tPA exposure (A,B) and with tPA exposure (C,D) at magnifications (10× and 40×, respectively). Clot containing fibrinogen-bio with FND (green NVN)-avidin without tPA exposure (E,F) and with tPA exposure (G,H) at magnifications 10× and 40×, respectively. Scale bars are 100 µm for 10× and 50 µm for 40×. Ex/Em: 470/488LP.
Figure 5
Figure 5
(A) Image of FITC-labeled clot at 10× prior to investigation at higher magnification. (B) Image of FITC-labeled clot after viewing several areas at higher magnification (40×) and then reimaging at 10×, showing the appearance of “holes” formed due to FITC bleaching (stars). Scale bars 100 µm. Ex/Em: 470/488LP.
Figure 6
Figure 6
Comparison of photobleaching under different labeling conditions for organic fluorophores as compared to FND fluorophores for red and green emission. (A–D) Avidin fluorophores labeling biotinylated CD41 at 100×, labeling activated platelets within the clot. Phycoerythrin-avidin-labeled activated platelets are not visible after 30 s (A,B), whereas FND (red NV) does not change (C,D). E-H show labelled fibrinogen at 40×. FITC shows dramatic reduction in intensity over 30 s (E,F), whereas FND (green NVN) does not discernably change (G,H). Green Ex/Em: 470/488LP; red Ex/Em: 517/561LP.
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