The novel AS system comprises a light source (CTV-A, Olympus Corporation, Tokyo, Japan), an angioscope of the monorail type (VecMover, Clinical Supply Co., Gifu, Japan), and a color chilled charged device (CCD) camera (CSVEC-10, Clinical Supply) (Fig. 1) [11].

After coronary angiography, the angioscope is introduced into the targeted coronary artery. The balloon of the angioscope is inflated to stop the blood flow therein, and the fiberscope incorporated into the angioscope is slowly advanced up to 7 cm distally to facilitate successive observations of the artery while displacing the blood by infusion of heparinized saline solution (10 IU/mL) at a rate of 2 mL/s for 10–20 s through the flash channel of the angioscope. To accurately confirm the location of the angioscope tip (and accordingly the observed portion), the angioscopic and fluoroscopic images are displayed simultaneously on a monitor.

After the control observation, 1 mL of 2.5 % EB solution is injected during balloon inflation into the artery through the flush channel of the angioscope to stain damaged endothelial cells or fibrin, and then, the balloon is deflated to restore blood flow. At 1–2 min later, the balloon is re-inflated and the coronary luminal surface is observed by AS [12].

Coronary ECs protect the vascular wall against spasm and thrombus formation through release of vasodilating and antithrombotic substances. When the ECs are damaged, thrombus immediately forms on that site [3]. However, it has been difficult to visualize damaged ECs in vivo.

Using dye-staining AS, damaged coronary ECs can be visualized, and it has become clear that cell damage is caused by insertion of a catheter for percutaneous intervention or balloon inflation and even by insertion of a guidewire (Fig. 2) [3, 13].

Platelet thrombi play a key role in the genesis of acute coronary syndrome (ACS), and it has been generally considered that white thrombus is platelet thrombus [3]. Using dye-staining coronary AS and EB as a marker of fibrin to examine whether white coronary thrombus in patients with ACS is composed of platelets alone, it was revealed that the majority of white thrombi (so-called platelet thrombi) can be clearly discriminated into fibrin-rich and platelet-rich thrombi (Fig. 3) [12]. Therefore, the use of this imaging modality may contribute to selection of effective primary or adjunctive thrombolytic therapy.

To date, there has not been an established imaging method to identify platelet thrombus in patients. My group found that fluorescein stains both fibrin and platelets (unpublished observation), so administering fluorescein after staining fibrin with EB may contribute to identification of platelet thrombus.

After confirming the existence of a thrombus in the culprit coronary segment in patients with ACS, EB is injected into the coronary artery. Next, 1 mL of 1 % solution of fluorescein is injected into the same artery, and the fluorescence is imaged with a fluorescent AS system using a band-pass filter of 470 nm and band-absorption filter of 515 nm. Details are described elsewhere [11].

Following fluorescein injection, the white portion of the thrombus, which does not stain with EB, exhibits fluorescence, indicating that it consists of platelets (Fig. 4).

Figure 5 shows a patient with unstable angina (UA) in whom the left anterior descending artery was totally occluded according to angiography. However, a residual lumen was observed and nothing was seen in the lumen by conventional AS. Dye-staining AS exposed a blue structure occupying the residual lumen, indicating a fibrin thrombus causing total occlusion. Such transparent fibrin thrombi, namely a structure that is not visible with conventional AS and becomes visible with dye-staining AS, have been observed in patients with UA or non-ST elevation myocardial infarction (non-STEMI) but not in those with STEMI [14•].

Coronary in-stent thrombosis is not infrequently observed by AS, even 6 months or more after stenting, despite the use of ticlopidine and aspirin. However, the mechanisms underlying late thrombosis are not well understood. ECs are highly antithrombotic, so it is possible that the mechanism is damage of the neo-ECs covering the stent struts.

Angioscopic observation of the coronary segments 6 months after bare stent implantation was performed in 44 patients. Stent struts were classified by conventional AS into subgroups: not covered by neointima (naked group); visible through the neointima (see-through group); and not visible through the neointima (not-see-through group). EC damage visualized with EB was observed in 13.3 % of the not-see-through group and in 80 % of the see-through/naked group. The neo-ECs covering stent struts in the see-through/naked group stained blue with EB more frequently than in the not-see-through group, indicating neo-EC damage (Fig. 6), which was classified as localized or diffuse damage. Late stent thrombosis was observed in the latter type. In animals, late stent thrombosis occurs when the neointimal thickness is within 100 μm. Neo-ECs may be damaged by friction between them and the stent struts, because of a thin interposed neointima, which should act as a cushion, resulting in late stent thrombosis [15, 16]. These findings suggest the necessity of an appropriate thickness of neointima for prevention of late stent thrombosis. A similar mechanism may cause late stent thrombosis in patients in whom a drug-eluting stent is implanted.

There are a considerable number of patients with ACS in whom no significant coronary obstruction is angiographically demonstrable. Hitherto, coronary spasm or accidental thrombosis has been considered as the underlying mechanism but without definite evidence. My group noticed a certain group of patients with ACS in whom significant stenosis is not demonstrable, and the suspect culprit coronary segment exhibits a “fluffy” (or frosted glass-like) surface. In a few of these patients, a thrombus distal to the fluffy segment is detected. The fluffy surface stains blue with EB, indicating the presence of fibrin and/or damaged ECs (Fig. 7).

Similar changes were reproduced by mechanical damage to the artery followed by blood perfusion to produce thrombus in dogs. Fluffy surface was exposed after removal of the globular thrombus. Histologically, the fluffy surface was composed of fibrin threads arising from the damaged ECs and adhered together by platelets. Therefore, it is proposed that a coronary segment with a fluffy or frosted glass-like surface is the site of surface disruption, and resultant thrombosis and the changes are related to residual fibrin and platelets after autolysis of the thrombus [17].

Deployment of bare metal or drug-eluting stents into the coronary artery is widely performed to treat coronary artery disease (CAD). However, it has become evident that this effective therapeutic modality brings about the occurrence of several unwanted phenomena in patients, for instance in-stent restenosis and subacute or late stent thrombosis.

In the course of conducting routine evaluations of implanted stents using coronary AS, I and my colleagues frequently found band-like structures connecting two or more stent struts, namely a web and/or membrane-like structure connecting multiple stent struts like a curtain and obstructing the lumen. The incidence of this phenomenon was further investigated in the acute and chronic phases of the deployment of stents in patients suffering from ACS and from effort angina pectoris. Additional evaluation experiments were conducted in animals to obtain further clarification of the mechanisms entailed in this phenomenon.

Web and membrane were observed in both patients with ACS and those with effort angina (80.0 vs 18.7 %) immediately after stent deployment and also 6 months later (55.5 vs 28.5 %). The structures stained blue with EB immediately after stent deployment but not 6 months later. In beagles, the web and membrane were observed on stent edges in 75.0 % of cases at 5 h after stenting and in 66.6 % cases at 1 month later. Histological studies revealed that the web and membrane examined 5 h after stenting were composed of fibrin, whereas those examined 1 month later were composed of collagen fibers (Fig. 8) [18].

The results indicated that web and membrane frequently form on the edges of coronary stents and in the acute phase are composed of fibrin, which is replaced by collagen fibers in chronic phase. It is conceivable that these structures form because of blood flow turbulence and the affinity of stent struts for platelets/fibrin.

Those findings also indicated that pretreatment with heparin is not sufficient for prevention of web and membrane formation. Therefore, prophylactic fibrinolytic therapy just before stent deployment may be necessary to prevent this unwanted phenomenon.

Slow-flow phenomenon occurs in the stented coronary artery. Distal embolism and EC dysfunction have been proposed as the main causative mechanisms of this unwanted hemodynamic change [19]. Also, stent edge restenosis occurs in chronic phase [20]. There is a possibility that in addition to these mechanisms, the formation of web and membrane on the stent edges also contributes to the slow or no-flow phenomenon and stent edge restenosis.

The CS system comprises a light source (CLV-A, Olympus), 9-F guiding balloon catheter (Clinical Supply), 4.2-F fiberscope (AF 14, Olympus), and a color CCD camera (OTV-A, Olympus) (Fig. 10).

Patients are pretreated with oral diazepam (10 mg) before being transferred to the catheterization laboratory. Left ventriculography is performed after administering 50 mg of intravenous lidocaine and 5,000 IU heparin. A 9-F guiding balloon is then introduced into the left ventricle and the balloon inflated with CO₂. Next, a 5-F fiberscope is advanced through the catheter to position the fiberscope tip at the tip of the catheter. The balloon is gently pushed against the targeted wall segment of the left ventricle, and 50–100 mL of saline solution (heparin 10 IU/mL, 37 °C) is injected through the catheter at 10 mL/s to displace the blood between the balloon and the ventricular endocardial surface being observed. The anterior, apical, inferior, and lateral walls of the left ventricle are observed. The changes in the endocardial surface are recorded on a DVD recorder using the color CCD camera.

After the control observation, the balloon catheter is replaced by a Judkins catheter, and 1 mL of 2.5 % EB is injected into the coronary artery that irrigates the left ventricular wall segment under study. The balloon catheter and fiberscope are reintroduced into the left ventricle, placing the balloon catheter tip on the same wall segment that was previously observed, and the luminal surface changes are observed again [9].

The observed portion is the anteroapical segment in chest pain syndrome; the wall segment that is irrigated by the artery in which spasm is evoked by intracoronary administration of acetylcholine in vasospastic angina (VSA); the wall segment irrigated by the stenotic artery in angina; and the dys- or akinetic wall segment in old myocardial infarction.

In patients without CAD, the endocardial surface diffusely stains blue immediately after intracoronary injection of EB, indicating normal SMBF in all patients with chest pain syndrome. In contrast, in patients with organic CAD, patchy staining indicating patchy preservation of SMBF and no staining indicating absent SMBF are frequently observed. In addition, patchy staining is also observed in patients with VSA, a functional disease (Fig. 11).

The effects of coronary stenting on SMBF have also been investigated, confirming restoration of SMBF by dye-staining CS in selected patients [9].

MTFF plays a critical role in maintaining the function and survival of cardiomyocytes. The myocardial tissue fluid receives oxygen and nourishments from the coronary arterial capillaries and conveys them to the cardiomyocytes, receiving carbon dioxide and waste matter from the cardiomyocytes and conveying them to the venous capillaries. Therefore, it is important to measure the MTFF, but there are no clinically available tools to measure it in vivo.

Fluorescein is used clinically to evaluate retinal vessels [34]. Within the blood vessel, fluorescence of this dye is masked by the corpuscles, but when separated from these, the dye exhibits strong fluorescence [3]. When injected into the coronary vessels, it diffuses through the microvascular wall, separate from the blood corpuscles, into the interstitial spaces and exhibits strong fluorescence, representing tissue fluid flow. Therefore, if the fluorescence of this dye in the myocardium can be visualized in vivo, a real-time evaluation of MTFF can be attained.

My group developed a fluorescent CS system, and using fluorescein as an indicator, the left ventricular subendocardial MTFF was evaluated in patients with CAD. Furthermore, the effects on MTFF of percutaneous coronary intervention or vasodilating agents were evaluated.